MD Virginia Boccardi

MD Immacolata Ambrosino

MD Michela Papa

MD Daniela Fiore

MD Maria Rosaria Rizzo

Professor Giuseppe Paolisso
email: giuseppe.paolisso@unina2.it

MD. Michelangela Barbieri

POTENTIAL ROLE OF TCF7L2 GENE VARIANTS ON CARDIAC SYMPATHETIC/PARASYMPATHETIC ACTIVITY

Keywords:

Variants in Transcription Factor 7-like 2 gene (TCF7L2) have been found strongly associated with an increased risk of type 2 diabetes as well as with an impairment of GLP-1 (glucagon like peptide-1) signalling chain. In rats, stimulation of central GLP-1 receptors increases heart rate and activates autonomic regulatory neurons. We aimed to evaluate the potential role of TCF7L2 gene polymorphisms on sympathovagal response in relation to changes in plasma insulin and/or GLP-1 concentration following glucose ingestion. Genotyping was performed for rs12255372 and rs7903146 TCF7L2 gene variants in 250 non related healthy volunteers (mean age 27 ± 3 yrs). Consistent with previous reports, both SNPs were in strong linkage disequilibrium (D'=0.87, r 2 =0.76). A subset of 167 patients underwent an oral glucose tolerance test while a continuous recording of Heart Rate Variability was performed. At baseline, no difference in fasting plasma insulin, in GLP-1 levels and in LF/HF ratio between the three genotypes were found. Along with glucose ingestion TT subjects had lower INS AUC (insulin area under curve) as well as higher LF/HF AUC (low frequency/high frequency area under curve) values. No difference in GLP-1 AUC (GLP-1 area under curve) between TCF7L2 gene variants was found. A multivariate analysis including multiple covariates showed that only INS AUC , GLP1 AUC and TCF7L2 gene variants were independently associated with LF/HF AUC . In conclusion TT genotype of rs12255372 and rs7903146 TCF7L2 gene variants is associated with lower insulin secretion and higher cardiosympathetic activity. Moreover such effect is independent of GLP-1 and insulin plasma concentrations suggesting a potential role of such gene variants in increasing cardiovascular risk via enhanced sympathetic nervous system activity.

INTRODUCTION

TCF7L2 plays a role in the wingless-type MMTV integration site family, member 1 (WNT) signalling pathway which influence the synthesis, and possibly secretion from intestinal L cells, of GLP1, an incretin hormone with insulinotropic and insulino-mimetic actions regulating glucose and energy homeostasis, food intake and cardiovascular functions [START_REF] Jin | The Wnt signalling pathway effector TCF7L2 and Type II diabetes mellitus[END_REF][START_REF] Yu | Antihypertensive effect of glucagon-like peptide 1 in Dahl salt-sensitive rats[END_REF].

Variants in the transcription factor 7-like 2 (TCF7L2) gene have been found strongly associated with an impaired insulin secretion and with an increased risk of type 2 diabetes (T2D) [START_REF] Grant | Variant of transcription factor 7-like 2 (TCF7L2) gene confers risk of type 2 diabetes[END_REF][START_REF] Groves | Association analysis of 6,736 U.K. subjects provides replication and confirms TCF7L2 as a type 2 diabetes susceptibility gene with a substantial effect on individual risk[END_REF][START_REF] Florez | TCF7L2 polymorphisms and progression to diabetes in the Diabetes Prevention Program[END_REF][START_REF] Damcott | Polymorphisms in the transcription factor 7like 2 (TCF7L2) gene are associated with type 2 diabetes in the Amish: replication and evidence for a role in both insulin secretion and insulin resistance[END_REF][START_REF] Saxena | Common single nucleotide polymorphisms in TCF7L2 are reproducibly associated with type 2 diabetes and reduce the insulin response to glucose in nondiabetic individuals[END_REF][START_REF] Zhang | Variant of transcription factor 7-like 2 (TCF7L2) gene and the risk of type 2 diabetes in large cohorts of U.S. women and men[END_REF][START_REF] Cauchi | Transcription factor TCF7L2 genetic study in the French population: expression in human beta-cells and adipose tissue and strong association with type 2 diabetes[END_REF][START_REF] Scott | Association of transcription factor 7-like 2 (TCF7L2) variants with type 2 diabetes in a Finnish sample[END_REF][START_REF] Schafer | Impaired glucagons-like peptide-1induced insulin secretion in carriers of transcription factor 7-lije 2 (TCF7L2) gene polymorphisms[END_REF].

Shafer et al. [START_REF] Schafer | Impaired glucagons-like peptide-1induced insulin secretion in carriers of transcription factor 7-lije 2 (TCF7L2) gene polymorphisms[END_REF] have recently showed that the presence of T allele of rs7903146 and rs12255372 TCF7L2 gene single-nucleotide polymorphisms (SNPs) was associated with reduced GLP-1-induced insulin secretion probably due to an impairment of GLP-1 signalling chain [START_REF] Yu | Antihypertensive effect of glucagon-like peptide 1 in Dahl salt-sensitive rats[END_REF][START_REF] Schafer | Impaired glucagons-like peptide-1induced insulin secretion in carriers of transcription factor 7-lije 2 (TCF7L2) gene polymorphisms[END_REF].

Interestingly, previous animal data have revealed beneficial effects of GLP-1 and its analogues on cardiovascular system by improving endothelial function and increasing myocardial contractility [START_REF] Yu | Antihypertensive effect of glucagon-like peptide 1 in Dahl salt-sensitive rats[END_REF][START_REF] Grant | Variant of transcription factor 7-like 2 (TCF7L2) gene confers risk of type 2 diabetes[END_REF][START_REF] Groves | Association analysis of 6,736 U.K. subjects provides replication and confirms TCF7L2 as a type 2 diabetes susceptibility gene with a substantial effect on individual risk[END_REF][START_REF] Florez | TCF7L2 polymorphisms and progression to diabetes in the Diabetes Prevention Program[END_REF][START_REF] Damcott | Polymorphisms in the transcription factor 7like 2 (TCF7L2) gene are associated with type 2 diabetes in the Amish: replication and evidence for a role in both insulin secretion and insulin resistance[END_REF][START_REF] Saxena | Common single nucleotide polymorphisms in TCF7L2 are reproducibly associated with type 2 diabetes and reduce the insulin response to glucose in nondiabetic individuals[END_REF][START_REF] Zhang | Variant of transcription factor 7-like 2 (TCF7L2) gene and the risk of type 2 diabetes in large cohorts of U.S. women and men[END_REF][START_REF] Cauchi | Transcription factor TCF7L2 genetic study in the French population: expression in human beta-cells and adipose tissue and strong association with type 2 diabetes[END_REF][START_REF] Scott | Association of transcription factor 7-like 2 (TCF7L2) variants with type 2 diabetes in a Finnish sample[END_REF][START_REF] Schafer | Impaired glucagons-like peptide-1induced insulin secretion in carriers of transcription factor 7-lije 2 (TCF7L2) gene polymorphisms[END_REF][START_REF] Nystrom | Effects of glucagon-like peptide-1 on endothelial function in type 2 diabetes patients with stable coronary artery disease[END_REF][START_REF] Ban | Cardioprotective and vasodilatory actions of glucagon-like peptide 1 receptor are mediated through both glucagon-like peptide 1 receptor-dependent and -independent pathways[END_REF]. Moreover, GLP-1 is a neuropeptide synthesized by neurons in the caudal regions of the nucleus of the solitary tract, and GLP-1 receptors are also found in the heart and in areas of the central nervous system that govern autonomic control [START_REF] Merchenthaler | Distribution of pre-pro-glucagon and glucagon-like peptide-1 receptor messenger RNAs in the rat central nervous system[END_REF][START_REF] Wei | Tissue-specific expression of the human receptor for glucagonlike peptide-I: brain, heart and pancreatic forms have the same deduced amino acid sequences[END_REF].

In rats, stimulation of central GLP-1 receptors increases blood pressure and heart rate and activates autonomic regulatory neurons [START_REF] Yamamoto | Glucagon-like peptide-1 receptor stimulation increases blood pressure and heart rate and activates autonomic regulatory neurons[END_REF][START_REF] Yamamoto | Glucagon-like peptide-1-responsive catecholamine neurons in the area postrema link peripheral glucagon-like peptide-1 with central autonomic control sites[END_REF]. In humans, only few data have been provided, showing that GLP-1 increases muscle sympathetic nerve traffic without affecting heart rate, plasma catecholamines, or cardiovagal and cardiosympathetic activity [START_REF] Bharucha | Effects of glucagon-like peptide-1, yohimbine, and nitrergic modulation on sympathetic and parasympathetic activity in humans[END_REF].

The potential role of TCF7L2 gene polymorphisms on sympathovagal response in relation to changes in plasma insulin and/or GLP-1 concentration following glucose ingestion is still unknown. In healthy young subjects, glucose ingestion and insulin infusion increases LF/HF ratio, an index of cardiac sympathovagal balance [START_REF]Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology Heart rate variability. Standards of measurements, physiological interpretation, and clinical use[END_REF][START_REF] Malliani | Cardiovascular neural regulation explored in the frequency domain[END_REF][START_REF] Paolisso | Glucose ingestion affects cardiac ANS in healthy subjects with different amounts of body fat[END_REF], producing a shift in cardiac autonomic nervous system activity toward sympathetic predominance [START_REF] Paolisso | Glucose ingestion affects cardiac ANS in healthy subjects with different amounts of body fat[END_REF][START_REF] Paolisso | Effects of different insulin infusion rates on heart rate variability in lean and obese subjects[END_REF].

Indeed, an alteration in the WNT signalling pathway related to TCF7L2 gene variant might result in an altered GLP-1 response and/or insulin secretion, which in turn could lead to an impaired cardiac sympathovagal balance an thus to an increased cardiovascular risk.

Thus, our study aims to: a) verify the effects of TCF7L2 gene polymorphisms on baseline and glucose ingestion stimulated plasma insulin and GLP-1 levels; b) evaluate the potential role of TCF7L2 gene variants in regulating cardiac sympathetic activity at baseline and after glucose ingestion.

MATERIALS AND METHODS

Subjects

Two hundred and fifty Caucasians non related non-diabetic subjects (115 men and 135 women), from 21-35 years of age (mean age 28 ± 3), living in Southern Italy, volunteered for the study. Clinical information was obtained by routine laboratory analyses, history and physical examination. All subjects were not hypertensive, had no evidence of metabolic or cardiovascular diseases and had liver, kidney, and thyroid function tests within the normal range. According to American Diabetes Association criteria, no subject was diabetic or affected by impaired fasting glucose [START_REF] Aberti | Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus. Provisional report of WHO consultation[END_REF]. The participants did not take any drugs known to affect glucose tolerance. After a clear explanation of the potential risk of the study, all subjects provided written informed consent to participate in the study, which was approved by the Ethical Committee of our Institutions.

Analytical methods

Anthropometric determinations (weight, height and BMI) were measured by standard technique. Body mass index (BMI) was calculated as weight in kilograms divided by square of height expresses in meters (Kg/m 2 ). Waist circumference was measured at the midpoint between the lower rib margin and the iliac crest and hip circumference at the level of the grater trochanter. Waist/hip ratio was calculated. Plasma glucose was determined immediately by glucose oxidase method (Glucose Autoanalayzer, Beckman Coulter, Inc., Fullerton, CA).

Plasma fasting cholesterol and triglycerides were determined by routine laboratory methods (Roche Diagnostics, GmbH, Mannheim, Germany).

For estimation of circulating intact GLP-1, plasma immunologic active forms of GLP-1 was determined using a specific ELISA kit (LINCO Research U.S.A.). Active isoforms of GLP-1 include GLP-1(7-36) amide and glycine-extended GLP-1(7-37). After secretion from enteroendocrine L cells, GLP-1 amide is rapidly degraded by dipeptidyl peptidase-4 (DPP-4) to its N-terminally truncated metabolite GLP-1(9-36), which does not interact with the known GLP-1 receptor. Blood samples were collected in ice-cooled EDTA-plasma tubes and immediately, within 30 seconds, was added appropriate amount of DPP-IV inhibitor according to manufacture's directions. Tubes was centrifuged at 2500 rpm for 10 minutes in refrigerated centrifuge. Samples were stored at -80° C. Plasma insulin quantitative determinations were obtained using Mercodia Insulin ELISA kit.

Genotyping

A total of 250 individuals were genotypized for rs12255372 (intron 3) G/T and rs7903146 (intron 3 C/T) allelic variants of TCF7L2 gene on chromosome 10q25.3 (Genbankgi: 89161187). DNA was isolated from whole blood collected into EDTA-containing tubes using DNA extraction kits (Illustra Blood GenomicPrep Mini Spin Kit, GE Healthcare). The rs12255372 polymorphism was identified using the following primers: upstream, 5'-GGC TTG CAG GTC AGA TTT TC-3', downstream 5'-ATT TGG CAT TCA AAT GGA GG-3'.

Polymerase chain reaction was carried out under the following conditions: 94°C for 4 minutes, followed by 35 cycles of 94°C for 30 seconds, 62°C for 30 seconds, 72°C for 30 seconds, with a final extension of 72°C for 10 minutes. Restriction fragment length polymorphism was carried out using Tsp509I enzyme. The resulting products were electrophoresed on a 4.5% agarose gel.

The rs7903146 was identified using the following primers: forward 5'-AAG AGA AGA TTC CTT TTT AAA TGG TG-3', reverse 5'-CCT CAT ACG GCA ATT AAA TTA TAC A-3'.

Polymerase chain reaction was carried out under the following conditions: 95°C for 5 minutes, followed by 34 cycles of 95°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds, with a final extension of 72°C for 9 minutes. Restriction fragment length polymorphisms was carried out using Hpy-CH4III enzyme. The resulting products were electrophoresed on a 4.5% agarose gel.

In order to better assess the potential role of TCF7L2 gene polymorphism on HRV and on glucose ingestion induced GLP-1 levels, 167 subjects underwent an oral glucose tolerance test (OGTT) while a continuous recording of HRV by the Holter technique was performed.

OGTT

A subset of 167 patients underwent an oral glucose tolerance test (OGTT) for stimulating a secretion patterns of GLP-1. Tests were performed after an overnight fast of 12 h. At 08:00 hours, participants ingested, within five minute, a solution containing 75 g anhydrous glucose dissolved in 250 ml water. Venous blood samples were obtained at 0', 30', 60', 120' and 180'min for determination of plasma glucose, insulin and GLP-1 concentrations. All subjects were studied in a comfortable supine position.

Cardiovascular determinations

All cardiovascular measurements were carried out under quiet conditions in a room maintained at 21°C., in a supine position for at least 30 min before starting the baseline Holter recording. Subjects with a respiratory rate less than 10 breaths/min (i.e. <0.15 Hz) were excluded from the study, in order to avoid the overlap of oscillations of low and high frequency at spectral analysis. Blood pressure and heart rate were recorded starting at 08.00 hours for a 120 min period (30 min baseline and 90 min after glucose ingestion) by using respectively a standard mercury sphygmomanometer and a 2channel 24-hour Holter ECG recordings (Del Mar Medical Systems, Irvine, Calif). All recordings were visually examined and manually over read to verify beat classification.

Abnormal beats and areas of artifact were automatically and manually identified and excluded from the analysis. The frequency domain analysis of HRV was performed [START_REF] Kleiger | Heart rate variability: measurement and clinical utility[END_REF]. The mean heart rate was calculated, for a complete 10-minute segment of RR interval, 10 min before and 10 min after glucose ingestion (T0), for the time 20 to the time 30 (T1), for the time 50 to the time 60 (T2), for the time 110 to the time 120 (T3). Spectral components were identified and estimated by using the spectral decomposition algorithm proposed by Johnsen and Andersen and were then assigned [START_REF] Jonsen | On power estimation in maximum entropy spectral analysis[END_REF], on the basis of their central frequency, to 1 of the 3 bands: very-low-frequency (VLF) (0-0.03 Hz), LF (0.04-0.15 Hz), and HF (0.16-0.45 Hz) [START_REF]Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology Heart rate variability. Standards of measurements, physiological interpretation, and clinical use[END_REF]. The low (LF) and high (HF) frequency components are normally considered [START_REF]Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology Heart rate variability. Standards of measurements, physiological interpretation, and clinical use[END_REF] and reported in normalized units [START_REF] Malliani | Cardiovascular neural regulation explored in the frequency domain[END_REF], which represent the relative value of the power of each component in proportion to the total power minus the VLF component. Because low-frequency power (LF) is modulated by baroreceptors and reflects both sympathetic and parasympathetic influences and highfrequency power (HF) reflects parasympathetic (vagally mediated) respiratory variation, LF/HF ratio, rather than LF, reflect relative sympathetic-parasympathetic activity and was used for calculating and reporting our data.

Calculations and statistical analysis

All data are presented as means ± SD. Areas under the curve for insulin (INS AUC ), glucose (GLY AUC ) and GLP-1 (GLP-1 AUC ) were calculated by trapezoidal method. To approximate normal distribution, plasma insulin and triglycerides were log-transformed for data analyses and back transformed for data presentation. Genotype frequencies were compared for investigating the Hardy-Weinberg equilibrium model by the χ2 test. One-way ANOVA followed by Bonferroni multiple testing correction was applied to assess differences in clinical, biochemical and cardiovascular data among the genotype groups. Simple correlation analyses allowed us to study the relationship between variables. Multivariate GLM (General Lineal Model) analyses were used to evaluate the difference in plasma glucose and triglycerides among different genotype, after adjustment for gender, BMI and WHR. A multivariate linear regression analyses allowed us to investigate the association of TCF7L2 gene variants with LF/HF ratio independently of plasma GLP-1 levels, BMI, gender, INS AUC and GLY AUC . A P < 0.05 was chosen for levels of significance. Statistical analyses were performed using SPSS 15 software package (SPSS, Inc., Chicago, IL).

In order to investigate the difference in cardiovascular and metabolic parameters between the two genotype groups, sample size calculation was estimated on an IBM PC computer by GPOWER software. The resulting sample size, estimated according to a global effect size of 27 % with type I error of 0.05 and a power of 80% was 110 patients. Pairwise LD coefficients (D') between SNPs were calculated by Haploview software version 4.0.

RESULTS

Clinical Data

Clinical and biochemical characteristics of the study subjects are reported in Table 1. All subjects (n=250) were young (mean age= 28.1±3.2), not obese, with no difference in gender ratio (135F/115M, χ2=1.6 p=0.206). According to gender ratio, female had a significantly lower BMI (21.8±2.27 vs 25.5±3.2 Kg/m 2 ; p < 0.001), WHR (0.75±0.08 vs 0.85±0.07; p < 0.001), plasma triglycerides (79.1±12 vs 99.7±27.2 mg/dl; p < 0.001) levels and higher HDLcholesterol (48.8±2.2 vs 46.6 ± 2.2 mg/dl; p< 0.001) levels compared to male subjects.

TCF7L2 gene variant analysis

The observed genotypes and allele distributions were in Hardy-Weinberg equilibrium (p>0.05). The T allele frequencies was 0.41 for rs12255372 and 0.39 for rs7903146. The frequency of rs12255372 GG, GT and TT genotypes was 32.3%, 53.2% and 14.5% respectively. As far as rs7903146 gene variant is concerned the frequency of CC, CT and TT genotypes was 35.4%, 48.8% and 15.8% respectively. The genotypic frequencies observed were almost similar to that previously reported in European populations and consistent with previous reports, both SNPs were in strong linkage disequilibrium (D'=0.87, r 2 =0.76). Because the two SNPs give the same information, results for SNP rs7903146 are shown in as supplemental material.

Table 1 shows clinical and biochemical characteristics of the whole population (n= 250) stratified according to rs12255372 (G/T) genotypes. No difference in genotype distribution between male and female subjects was found (χ 2 =1.115; p=0.573). Subjects carrying the TT genotype had significantly higher fasting plasma glucose levels compared to the GT and GG carriers, even after adjusting for gender, BMI and WHR (p=0.04). No differences in other clinical and biochemical parameters among the three genotypes were found.

Biochemical data and cardiovascular determinations over OGTT administration.

A subset of 167 subjects underwent an oral glucose tolerance test (OGTT) while a continuous recording of HRV by the Holter technique were performed. No subjects resulted affected by glucose intolerance.

At baseline, no significant correlations between LF/HF values and fasting plasma glucose (r=0.08, p= .397), insulin (r=0.12, p= .398) and GLP-1 (r=0.12, p= .691) levels were found.

In all subjects studied, oral glucose ingestion evoked a significant increase in plasma glucose (from 4.9 ±0.5 to 7.1±0.7 mmol/L p= 0.038) and insulin (from 49.6 ±8 to 118.3±10.2 pmol/L p= 0.01) levels at 60 minutes and in plasma GLP-1 (from 8.4 ±1 to 17.6±1 pmol/L p= 0.02) concentrations at 30 min compared to fasting values. The plasma peak of GLP-1 was transient and levels returned to near fasting within 90-120 minutes. Basal LF/HF ratio also rose significantly after 30 min (from 2.07 ±0.3 to 2.24 ±0.5 p=0.03 ) reaching the maximum peak at 60 min and later declined without significant difference vs baseline.

Categorizing subjects in different genotype groups, no difference in baseline plasma insulin, GLP-1 levels and in LF/HF ratio between genotypes for either SNP were found, while fasting plasma glucose levels were significantly higher in TT carriers (Fig. 1; Suppl. After glucose ingestion, no significant difference in plasma glucose and GLP1 levels between the three genotype groups for each SNP was found. Subjects with TT genotype for both rs12255372 and rs7903146 variants had a significantly lower plasma insulin levels at 60'and higher LF/HF ratio at 30' and 60 minutes. No difference in LF/HF ratio at baseline and after 120 minutes of glucose ingestion between the three genotype groups for either SNP was found No difference in GLP-1 AUC among TCF7L2 gene variants was found, while subjects carrying the TT genotype for both SNP had lower INS AUC as well as higher LF/HF AUC values compared the other genotype. No difference in INS AUC and LF/HF AUC between GG (or CC) and GT (or CT) genotype was also found. (Fig. 2; Suppl. Fig. 2)

The independent association of TCF7L2 gene variant with LF/HF ratio was tested in a multivariate analysis (Table 2). A model including LF/HF AUC , as the dependent variable and BMI, gender, GLP-1 AUC , GLY AUC , INS AUC , TCF7L2 (G/T) gene polymorphism as covariates showed that only INS AUC , GLP-1 AUC and TCF7L2 (G/T) gene variant were independently associated with LF/HF AUC .

DISCUSSION

The major findings of our investigation are: i) TT genotype of TCF7L2 variants is associated with lower insulin secretion and higher cardiosympathetic activity ii) TCF7L2 genotype effect on cardiosympathetic activity is independent of GLP-1 and insulin plasma concentrations.

TCF7L2 encodes a transcription factor playing an important role in the WNT signalling pathway [START_REF] Yi | TCF-4 mediates cell type-specific regulation of proglucagon gene expression by beta-catenin and glycogen synthase kinase-3beta[END_REF] required for the normal development of the pancreas and pancreatic islets and it is also involved in GLP-1 production [START_REF] Fehmann | Cell and molecular biology of the incretin hormones glucagon-like peptide-I and glucose-dependent insulin releasing polypeptide[END_REF]. Variants in TCF7L2 gene are strongly associated with an increased risk of type 2 diabetes (T2D) (1). Shafer et al. have recently demonstrated that the presence of the allele T of the rs12255372 TCF7L2 gene polymorphism is associated with reduced GLP-1-induced insulin secretion [START_REF] Schafer | Impaired glucagons-like peptide-1induced insulin secretion in carriers of transcription factor 7-lije 2 (TCF7L2) gene polymorphisms[END_REF].

Accordingly, in our study subjects carrying the TT genotype of TCF7L2 (G/T) gene had reduced insulin secretion. Interestingly, a predominant cardiosympathetic activity (higher LF/HF ratio) in response to glucose ingestion in TT subjects was also found. The higher LF/HF ratio found in TT carriers might contribute to explain the association of TCF7L2 gene polymorphism with an increased risk of type 2 diabetes. In fact, it is generally accepted that impaired autonomic function is not only the consequence, but also a precursor of hyperglycemia [START_REF] Bharucha | Effects of glucagon-like peptide-1, yohimbine, and nitrergic modulation on sympathetic and parasympathetic activity in humans[END_REF][START_REF] Seals | Chronic sympathetic activation: consequence and cause of ageassociated obesity?[END_REF]. Chronically elevated levels of sympathetic activity may lead to gradual down regulation of the beta-adrenoceptor-mediated thermogenic and metabolic responses, which may be involved in the development of obesity and insulin resistance as seen in new onset type 2 diabetes mellitus [START_REF] Bharucha | Effects of glucagon-like peptide-1, yohimbine, and nitrergic modulation on sympathetic and parasympathetic activity in humans[END_REF][START_REF] Seals | Chronic sympathetic activation: consequence and cause of ageassociated obesity?[END_REF][START_REF] Van Baak | Meal-induced activation of sympathetic nervous system and its cardiovascular and thermogenic effects in man[END_REF]. Moreover, our results might also indicate TCF7L2 polymorphism as a new potential cardiovascular risk factor. In fact, an unbalanced sympathetic/parasympathetic tone, with a prevalence of sympathetic activity, is associated with higher cardiovascular mortality in type 2 diabetic patients [START_REF] Tsuji | Reduced heart rate variability and mortality risk in an elderly cohort. The Framingham Heart Study[END_REF][START_REF] Mancia | The sympathetic nervous system and the metabolic syndrome[END_REF].

Sympathetic nervous system is an important regulatory mechanism of metabolic and cardiovascular functions [START_REF] Reaven | Hypertension and associated metabolic abnormalities--the role of insulin resistance and the sympathoadrenal system[END_REF]. In healthy young subjects, glucose ingestion enhances LF/HF ratio, which is related to the insulin sensitivity, and negatively correlated with body fat content [START_REF] Paolisso | Glucose ingestion affects cardiac ANS in healthy subjects with different amounts of body fat[END_REF]. It is well accepted that insulin stimulates sympathetic activity independently of changes in plasma glucose concentration [START_REF] Berne | The sympathetic response to euglycaemic hyperinsulinaemia. Evidence from microelectrode nerve recordings in healthy subjects[END_REF][START_REF] Anderson | Hyperinsulinemia produces both sympathetic neural activation and vasodilation in normal humans[END_REF] increasing the LF/HF ratio [START_REF] Schroeder | Diabetes, glucose, insulin, and heart rate variability: the Atherosclerosis Risk in Communities (ARIC) study[END_REF][START_REF] Manzella | Cardiac autonomic activity and Type II diabetes mellitus[END_REF]. Furthermore, in response to food intake (as well as glucose ingestion) intestinal enteroendocrine cells secrete multiple hormones, including Glucagon-like peptide-1 (GLP-1). GLP-1 given peripherally or centrally increases heart rate and blood pressure and activates autonomic regulatory neurons in rats thus suggesting that central GLP-1 regulates cardiovascular function by increasing the sympathetic system and inhibiting vagal activity [START_REF] Yamamoto | Glucagon-like peptide-1 receptor stimulation increases blood pressure and heart rate and activates autonomic regulatory neurons[END_REF][START_REF] Yamamoto | Glucagon-like peptide-1-responsive catecholamine neurons in the area postrema link peripheral glucagon-like peptide-1 with central autonomic control sites[END_REF].

Accordingly, in our study, the glucose induced increase in cardiosympathetic activity, was positively associated with increased insulin and GLP1 plasma levels. Furthermore, a multivariate analysis with LF/HF AUC as the dependent variable, confirmed an independent role of both insulin and GLP1 levels in stimulating the cardiosympathetic activity. Indeed, subjects carrying TT genotype had lower plasma insulin secretion and similar GLP1 levels compared to heterozygote and GG carriers. Furthermore, the multivariate analysis showed that TCF7L2 gene variant was significantly associated with AUC of LF/HF ratio independently of both insulin and GLP1 plasma levels, thus suggesting that an impaired GLP1 secretion is not likely to explain the increased sympathetic activity in carriers the risk allele. An altered GLP-1 signalling, rather than different GLP1 plasma levels, might explain the effect of TCF7L2 variant on cardio-sympathetic activity. Previous results, obtained by Shafer et al [START_REF] Schafer | Impaired glucagons-like peptide-1induced insulin secretion in carriers of transcription factor 7-lije 2 (TCF7L2) gene polymorphisms[END_REF], showing that, despite similar basal and glucose induced total plasma GLP1 levels, the presence of the allele T was associated with reduced insulin secretion following GLP-1 infusion compared to allele G, seem to support our hypothesis.

In our study the active form of GLP-1 (peptide 7-36 and 7-37), instead of the total GLP1 (peptide 9-36) was evaluated. Indeed, systemic GLP-1 levels may not adequately reflect the level of the active hormone at tissue level. In fact, impaired TCF7L2 activity, through the TT genotype, might tissue specifically reduce or increase the GLP1 levels. Thus, the reduced efficiency of GLP1 in stimulating insulin secretion in pancreatic beta cell and the increased 13 activity on cardiac autonomic system, found in our study, might both reflect a functional defect in GLP signal (ie an impaired GLP1 receptor function).

In conclusion, our study demonstrate that TT genotype of rs12255372 and rs7903146 TCF7L2 gene variants are associated with reduced insulin secretion and with increased cardio sympathetic activity. Such results might contribute to explain the association of TCF7L2 gene polymorphisms with an increased risk of type 2 diabetes and suggest a potential role of TCF7L2 gene variants in increasing cardiovascular risk via enhanced sympathetic nervous system (SNS) activity.

The limited number of subjects and the lack of replication study are potential limitations of this study. Thus, further studies will be necessary for replicating our findings in a larger population group, with ethnical diversity to better elucidate molecular mechanisms linking TCF7L2 gene and cardiac sympathetic/parasympathetic activity.

  Fig 1).

(Fig 1 ;

 1 Suppl Fig 1 ).AUC for insulin (INS AUC ), GLP-1(GLP-1 AUC ) and LF/HF (LF/HF AUC ) were also calculated. LF/HF AUC positively correlated with GLP-1 AUC (r= 0.39, p=0.03) and INS AUC (r= 0.48, p=0.03). A positive association between GLP-1 AUC and INS AUC (r= 0.52, p=0.02) was also found. All such association were still significant after adjusting for BMI (data not shown).

Disclosure statement :FIGURESFig. 1 .

 statement1 FIGURES

Fig. 2 .Fig. 1 .

 21 Fig. 2. LF/HF AUC , INS AUC and GLP-1 AUC values according to rs12233572 TCF7L2 gene polymorphism. Data are means ± standard errors. *p= 0.01 TT vs GT and TT vs GG by Bonferroni multiple testing correction.

  FIGURE 2

Table 1 . Clinical characteristics of study population according to genotype at the rs12255372

 1 TABLES values between groups, § p=0.01 TT vs GT; p=0.02 TT vs GG by one-way ANOVA followed by Bonferroni multiple testing correction.

		All	GG	GT	TT
		(n=250)	(n=81)	(n=133)	(n=36)	p
	Age (years)	28.1 ± 3.2 27.2 ± 3.4	27.6 ± 3.3	28.2 ± 2.7	.737
	Gender	135F/115M	42F/39M	73F/60M	20F/16M	.573
	BMI (Kg/m 2 )	23.2 ± 3.3 22.7 ± 3.3	23.9 ± 3.5	24.0 ± 2.8	.395
	WHR	0.77 ±0.12 0.76 ± 0.10	0.80 ± 0.11	0.78 ± 0.06	.196
	FPG (mmol/L)	5.1 ± 0.7	4.8 ± 0.4	4.7 ± 0.4	5.3 ± 0.4 §	.002
	TTotal cholesterol (mg/dl)	158 ± 17	161 ± 17	156 ± 17	158 ± 21	.567
	HDL cholesterol (mg/dl)	48 ± 2	48 ± 2	47 ± 3	48 ± 2	.455
	Triglycerides (mg/dl)	87 ± 21	87 ± 14	91 ± 23	98 ± 17	.321
	HR (beats/min)	71 ± 5	71 ± 6	72 ± 7	73 ± 6	.391
	SBP (mmHg)	113 ± 9	114 ± 10	111 ± 10	115 ± 10	.550
	DBP (mmHg)	72 ± 7	73 ± 8	71 ± 8	74 ± 7	.500
	Data are means ± standard deviation. BMI, Body Mass Index; WHR, Waist/Hip Ratio; FPG,
	fasting plasma glucose; HR, heart rate; SBP, systolic blood pressure; DBP, diastolic blood
	pressure. p

Table 2 : Linear multiple regression analyses with AUC of LF/HF ratio as the dependent variable

 2

	(n = 167)
		β	t	p
	Model 1
	BMI	-0.65	-0.621	.558	R 2 = .554
	Sex	0.03	0.027	.979
	AUC glucose
	(mmol/L x 180')	0.11	1.218	.259
	AUC insulin
	(pmol/L x 180')	0.32	1.041	.029
	AUC Glp1
	(pmol/L x 180')	0.18	1.953	.036
	TCF7L2
	polymorphism*	0.78	9.948	.022

BMI, Body Mass Index; AUC, area under curve;* Calculated as GG carrier (=1) vs GT carrier (=2) vs TT carrier (=3). β = standardized regression coefficient; t=" t "statistics computed by dividing the estimated value of the beta coefficient by its standard error.


Antonios Boultadakis

Nikolaos Pitsikas
email: npitsikas@med.uth.gr

EFFECTS OF THE NITRIC OXIDE SYNTHASE INHIBITOR L-NAME ON RECOGNITION AND SPATIAL MEMORY DEFICITS PRODUCED BY DIFFERENT NMDA RECEPTOR ANTAGONIST IN THE RAT

Keywords: L-NAME, MK-801, ketamine, recognition memory, spatial memory, rat C

There is consistent experimental evidence that non-competitive antagonists of the NMDA receptor, such as ketamine, MK-801, and phencyclidine (PCP) impair cognition and produce psychotomimetic effects in rodents. Nitric oxide (NO) is considered as an intracellular messenger in the brain. The implication of NO in learning and memory is well documented.

The present study was designed to investigate the ability of the NO synthase (NOS) inhibitor L-NAME to antagonize recognition and spatial memory deficits produced by the NMDA receptor antagonists, MK-801 and ketamine in the rat. L-NAME (1-3 mg/kg) counteracted MK-801 (0.1 mg/kg) and ketamine (3 mg/kg)-induced performance impairments in the novel object recognition task. L-NAME (10 mg/kg) attenuated ketamine (15 mg/kg)-induced spatial working memory and retention deficits in the radial water maze paradigm. L-NAME, applied at 3 mg/kg however, disrupted rodents' performance in this spatial memory task. The present findings indicate a) that L-NAME is sensitive to glutamate hypofunction produced by other than PCP NMDA antagonists such as MK-801 and ketamine and b) that L-NAME alone differentially affects rodents' spatial memory.

INTRODUCTION

Schizophrenia is a serious mental disorder that affects up to 1% of the population worldwide.

Cognitive deficits in schizophrenic patients are core features of the illness, and predict vocational and social disabilities for patients [START_REF] Freedman | Schizophrenia[END_REF]. Numerous studies have indicated that the function of the glutamatergic system, in particular N-methyl-D-aspartate (NMDA) receptors, might be compromised in schizophrenia. Exposure to non-competitive NMDA receptor antagonists like phencyclidine (PCP) or ketamine produces behavioral symptoms in healthy individuals that resemble both positive and negative symptoms of schizophrenia [START_REF] Javitt | Recent advances in the phencyclidine model of schizophrenia[END_REF][START_REF] Krystal | Sub-anesthetic effects of the noncompetitive NMDA antagonist, ketamine, in humans. Psychotomimetic, perceptual, cognitive and neuroendocrine responses[END_REF] and exacerbate symptoms in schizophrenic patients [START_REF] Malhotra | Ketamine-induced exacerbation of psychotic symptoms and cognitive impairment in neuroleptic-free schizophrenics[END_REF][START_REF] Lahti | Effects of ketamine in normal and schizophrenic volunteers[END_REF]. In addition, ketamine, PCP or MK-801 induce schizophrenia-like symptoms, including cognitive deficits, in rodents [START_REF] Tricklebank | The behavioural effects of MK-801: a comparison with antagonists acting non-competitively and competitively at the NMDA receptor[END_REF][START_REF] Verma | NMDA receptor antagonists impair prefrontal cortex function as assessed via spatial delayed alternation performance in rats: modulation by dopamine[END_REF][START_REF] De Lima | Pre-or posttraining administration of the NMDA receptor blocker MK-801 impairs object recognition memory in rats[END_REF]. Due to these psychotomimetic properties, NMDA receptor antagonists are widely used as models of schizophrenia in animals [START_REF] Geyer | Animal models of psychiatric disorders[END_REF].

Nitric oxide (NO), a soluble, short-lived and freely diffusible gas, is an important intracellular messenger in the brain [START_REF] Garthwaite | Glutamate nitric oxide and cell-cell signalling in the nervous system[END_REF]. Reportedly, NO is involved in the mechanisms of synaptic plasticity in the hippocampus [START_REF] O'dell | Tests of the roles of two diffusible substances in long-term potentiation: Evidence for nitric oxide as a possible early retrograde messenger[END_REF][START_REF] Haley | The role of nitric oxide in hippocampal long-term potentiation[END_REF] and plays an important role in cognition [START_REF] Prast | Nitric oxide as modulator of neuronal function[END_REF].

A plethora of experimental data indicates the implication of NO in schizophrenia [START_REF] Bernstein | The many faces of nitric oxide in schizophrenia. A review[END_REF]. In this context, it has been observed an abnormal distribution of nitrergic neurons in the frontal and temporal lobes of schizophrenic patients, which may reflect that the normal pattern of neuronal migration during development of the cerebral cortex may be affected in these patients [START_REF] Akbarian | Altered distribution of nicotinamide-adenine dinucleotide phosphate-diaphorase cells in frontal lobe of schizophrenics implies disturbances of cortical development[END_REF]. In addition, experimental evidence suggests that polymorphisms in the neuronal nitric oxide synthase (nNOS) gene are associated with schizophrenia and prefrontal cortex (PFC) function in schizophrenic patients [START_REF] Reif | A neuronal nitric oxide synthase (NOS-I) haplotype associated with schizophrenia modifies prefrontal cortex function[END_REF]. Contradictory results were reported however, concerning the levels of NO metabolites in the serum of schizophrenic patients. Either high [START_REF] Taneli | Serum nitric oxide metabolite levels and the effect of antipsychotic therapy in schizophrenia[END_REF][START_REF] Yilmaz | Increased levels of nitric oxide, cortisol and adrenomedullin in patients with chronic schizophrenia[END_REF] or low [START_REF] Ramirez | Low concentrations of nitrite and nitrate in the cerebrospinal fluid from schizophrenic patients[END_REF] concentrations of NO metabolites have been reported.

Since both alterations in the synaptic organization of the brain [START_REF] Roberts | Synaptic differences in the post-mortem striatum of subjects with schizophrenia: a stereological and utrastructural analysis[END_REF] and neurotransmitter deficits [START_REF] Costa | A GABAergic cortical deficit dominates sschizpphrenia pathophysiology[END_REF] are key features of schizophrenia, excess NO might contribute to disturbed neurocircuitry in this disease. Reportedly, exceeding NO concentrations are associated with neuronal damage [START_REF] Dawson | Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures[END_REF], mitochondrial dysfunction [START_REF] Das | Nitric oxide, free radicals and polyamines may have a role in membrane pathology of schizophrenia[END_REF] and impairment of the NMDA-receptor mediated neurotransmission (Clinton et al, 2003). As a whole, these data suggest that reduced NOS activity in schizophrenia may be neuroprotective. Therefore, efforts are made to develop NOS inhibitors as possible therapeutic tools for schizophrenia [START_REF] Bernstein | The many faces of nitric oxide in schizophrenia. A review[END_REF].

A number of animal studies have demonstrated that diverse NOS inhibitors antagonized PCP-induced psychotomimetic effects [START_REF] Johansson | Nitric oxide synthase inhibition blocks phencyclidine-induced behavioural effects on prepulse inhibition and locomotor activity in the rat[END_REF][START_REF] Klamer | The nitric oxide synthase inhibitor, L-NAME, block phencyclidine-induced disruption of prepulse inhibition in mice[END_REF][START_REF] Klamer | The neuronal selective nitric oxide synthase inhibitor, Nomega-propyl-L-arginine, blocks the effects of phencyclidine on prepulse inhibition and locomotor activity in mice[END_REF]Klamer et al, 2005a;Klamer et al, 2005b). In this context, it has been proposed that NOS inhibition selectively amends the behavioral effects of PCP and not those of MK-801 (Klamer et al, 2005a). In addition, that application of the non-selective NOS inhibitor L-NAME counteracted PCP-induced acquisition deficits in a spatial learning task in the rat has also been reported (Wass et al, 2006a;Wass et al, 2006b).

Under our experimental conditions, we have observed that L-NAME (1-3 mg/kg) antagonized delay-dependent deficits in the novel object recognition memory task in the rat [START_REF] Boultadakis | Effects of the nitric oxide synthase inhibitor L-NAME on different memory components as assessed in the object recognition task in the rat[END_REF]. At the moment, it is not clear if and how L-NAME is able to counteract recognition memory deficits produced by NMDA hypofunction. In this context, we have observed that two NMDA receptor blockers with different profile in terms of potency or pharmacokinetic properties, MK-801 and ketamine, disrupted animals' performance in the novel object recognition paradigm [START_REF] Pitsikas | Nitric oxide donor molsidomine attenuates psychotomimetic effects of the NMDA receptor antagonist MK-801[END_REF][START_REF] Pitsikas | Effects of sub-anesthetic doses of ketamine on rats' spatial and non-spatial recognition memory[END_REF].

Taken the above evidences into account, the first aim of our study was to evaluate the efficacy of L-NAME in antagonizing MK-801 and ketamine induced-performance deficits in a recognition memory task in the rat. For this purpose, the novel object recognition task was selected. This test is a non-rewarded paradigm based on the spontaneous exploratory behavior of rats [START_REF] Ennaceur | One-trial object recognition in rats and mice: Methodological and theoretical issues[END_REF]. Subsequently, the efficiency of L-NAME to counteract ketamineinduced detrimental effects on spatial memory was assessed by using a radial water maze task [START_REF] Pitsikas | Effects of the active constituents of Crocus sativus L., crocins, on recognition and spatial rats' memory[END_REF].

MATERIALS AND METHODS

Animals

Different groups of male (3-month-old) Wistar rats (Hellenic Pasteur Institute, Athens, Greece), weighing 250-300 g, were used in this study. The animals were housed in Makrolon cages (47.5 x 20.5 x 27 cm), three per cage, in a regulated environment (21±1° C, 50-55% relative humidity, 12-h/12-h light/dark cycle, white lights on from 0700 to 1900 hours), with free access to food and water. Experiments were conducted in the room where only these animals where housed, and took place during the light cycle between 1000 and 1400 hours.

Procedures involving animals and their care were conducted in conformity with the international guidelines in compliance with National and International laws and policies. (EEC Council Directive 86/609, JL 358, 1, December 12, 1987; NIH Guide for Care and Use of Laboratory Animals, NIH publication no. 1985.

Novel object recognition test

The test apparatus and procedure have been extensively described earlier [START_REF] Boultadakis | Effects of the nitric oxide synthase inhibitor L-NAME on different memory components as assessed in the object recognition task in the rat[END_REF]. Before testing, for 3 consecutive days, rats were allowed to explore the apparatus for 2 min. Testing consisted of a session of two 2-min trials. During the "sample" trial (T1), two identical samples (objects) were placed in two opposite corners of the apparatus in a random fashion. A rat was placed in the middle of the apparatus and was left to explore these two identical objects. After T1, the rat was put back in its home cage and an intertrial interval (ITI) was given. Subsequently, the "choice" trial (T2) was performed. During T2, a new object (N) replaced one of the samples presented in T1, therefore, the rats were re-exposed to two objects: the familiar (F) and the new (N).

The times spent by rats in exploring each object during T1 and T2 were recorded manually by using a stopwatch. From this measure a series of variables was then calculated: the total time spent in exploring the two identical objects in T1, and that spent in exploring the two different objects, (F) and (N) in T2. The discrimination between (F) and the (N) during T2 was measured by comparing the time spent in exploring the (F) with that spent in exploring the (N). As this time may be biased by differences in overall levels of exploration a discrimination index (D) was then calculated; D=N-F/N+F. D is the discrimination ratio and represents the difference in exploration time expressed as a proportion of the total time spent exploring the two objects in T2 [START_REF] Cavoy | Spatial but not object recognition is impaired by aging in rats[END_REF]. In addition, motor activity of each animal expressed as total number of steps during each trial was also recorded.

Radial water maze test

Spatial reference and spatial working memories were studied using an eight-arm radial water maze procedure modified from [START_REF] Jarrard | On the role of the hippocampal connections in the performance of place and cue tasks: comparisons with damage to hippocampus[END_REF] and [START_REF] Pitsikas | Deterioration of spatial and nonspatial reference and working memory in aged rats: protective effect of life-long calorie restriction[END_REF]. The test apparatus has been described before [START_REF] Pitsikas | Effects of the active constituents of Crocus sativus L., crocins, on recognition and spatial rats' memory[END_REF].

Each trial involved placing the rat in the water, facing the wall, at the end of one of the seven unbaited arms (start positions). Each rat was allowed 90 s to escape onto the platform. If it failed to escape within this time, it was guided to the platform. When the rat found the escape platform it was allowed to stay there for 30 s. Then the rat was placed again into the maze for its next trial, which used the same platform location (e.g., channel 1) and a different starting position as the previous trial. This was repeated until the rat found the platform or 90 s had elapsed whichever occurred first. Each animal received a daily session of 2 trials for 4 consecutive days.

Choices of arms (defined by whole body entry) were recorded manually for each trial. The order of entry into the maze arms was recorded so that the total number of errors could be calculated. Errors were quantified in terms of reference and working memory incorrect errors [START_REF] Jarrard | On the role of the hippocampal connections in the performance of place and cue tasks: comparisons with damage to hippocampus[END_REF]. Reference memory errors were the number of first entries into a channel which had never contained a platform. Working memory incorrect errors were the number of repeated entries into an arm which had never contained a platform (repeated entries into a reference memory arm thus, reflecting the combined effects of failures of both working and reference memory) [START_REF] Okaichi | Scopolamine impairs both working and reference memory in rats: a replication and extinction[END_REF]. These parameters were averaged for each daily block of trials and for each rat, whose daily performance was thus characterized.

On day 5, the rats performed a single spatial probe trial [START_REF] Gage | Spatial learning and motor deficits in aged rats[END_REF]. This trial consisted of removing the platform from the baited arm of the radial water maze and allowing the rat to swim for 60 s in search of it. The time spent in each of the eight channels of the pool was calculated as a percentage over 60 s.

Drugs

[L-NAME, (+)-MK-801 maleate (Sigma, St. Louis, MO, U.S.A.)] and ketamine hydrochloride were dissolved in saline (NaCl 0.9%) and administered intraperitoneally (i.p.) in a volume of 1 ml/kg. Doses of L-NAME were chosen on the basis of a study in which they were effective against learning impairments and did not produce adverse side effects [START_REF] Boultadakis | Effects of the nitric oxide synthase inhibitor L-NAME on different memory components as assessed in the object recognition task in the rat[END_REF]. The doses of MK-801 (0.1 mg/kg) and ketamine (3 mg/kg) were selected based on previous findings in which were found to impair rat performance in the novel object recognition task without producing side effects [START_REF] Pitsikas | Nitric oxide donor molsidomine attenuates psychotomimetic effects of the NMDA receptor antagonist MK-801[END_REF][START_REF] Pitsikas | Effects of sub-anesthetic doses of ketamine on rats' spatial and non-spatial recognition memory[END_REF]. For the radial water maze test, the dose of ketamine (15 mg/kg) was chosen based on a previous report in which was found to impair place learning in a water maze task [START_REF] Wesierska | Differential effects of ketamine on the reference and working memory versions of the Morris water maze task[END_REF]. For all studies, control animals received isovolumetric amounts of the vehicle (NaCl 0.9%).

Experimental protocol Experiment 1: effects of L-NAME in antagonizing MK-801-induced performance deficits in the novel object recognition task

Rats were randomly divided into eight experimental groups (10 rats per group) as follows:

vehicle+vehicle; vehicle+L-NAME 1 mg/kg; vehicle+L-NAME 3 mg/kg; vehicle+L-NAME 10 mg/kg; MK-801 0.1 mg/kg+vehicle; MK-801 0.1 mg/kg+L-NAME 1 mg/kg; MK-801 0.1 mg/kg+L-NAME 3 mg/kg; MK-801 0.1 mg/kg+L-NAME 10 mg/kg. To study the effects of compounds on post-training memory components (storage and/or retrieval) compounds were administered immediately after T1. It has been reported that treatment with MK-801 (0.1 mg/kg) induce hypermotility [START_REF] Verma | NMDA receptor antagonists impair prefrontal cortex function as assessed via spatial delayed alternation performance in rats: modulation by dopamine[END_REF][START_REF] Homayoun | Functional interaction between NMDA and mGlu5 receptors: effects on working memory, instrumental learning, motor behaviors and dopamine release[END_REF] and produce a sustained increase in stereotypy that peaked 40 min after injection and returned to baseline 2 h later [START_REF] Homayoun | Functional interaction between NMDA and mGlu5 receptors: effects on working memory, instrumental learning, motor behaviors and dopamine release[END_REF]. Therefore, it is quite possible that these factors may interfere with learning performance. There is also experimental evidence that recognition memory abilities in the young rat remained intact when a delay condition up to 6 h was used [START_REF] Bartolini | Aniracetam restores object recognition impaired by age, scopolamine and nucleus basalis lesions[END_REF]. In an attempt to avoid the possibility that rats' performance is influenced by these already mentioned side effects due to the treatment with MK-801, the 3-h ITI was selected.

Experiment 2: effects of L-NAME in antagonizing ketamine-induced performance deficits in the novel object recognition task

Rats were randomly divided into eight experimental groups (10 rats per group) as follows: vehicle+vehicle; vehicle+L-NAME 1 mg/kg; vehicle+L-NAME 3 mg/kg; vehicle+L-NAME 10 mg/kg; ketamine 3 mg/kg+vehicle; ketamine 3 mg/kg+L-NAME 1 mg/kg; ketamine 3 mg/kg+L-NAME 3 mg/kg; ketamine 3 mg/kg+L-NAME 10 mg/kg. To study the effects of compounds on post-training memory components (storage and/or retrieval) compounds were administered just after T1. For this study, the 1-h ITI has been selected since at this delay condition recognition memory is still intact in the vehicle-treated rat [START_REF] Bartolini | Aniracetam restores object recognition impaired by age, scopolamine and nucleus basalis lesions[END_REF] and impairments associated with treatment with ketamine (hypermotility, stereotypies, ataxia) [START_REF] Verma | NMDA receptor antagonists impair prefrontal cortex function as assessed via spatial delayed alternation performance in rats: modulation by dopamine[END_REF] were not observed [START_REF] Pitsikas | Effects of sub-anesthetic doses of ketamine on rats' spatial and non-spatial recognition memory[END_REF].

Radial water maze task

Preliminary results have shown that rats treated with L-NAME 3 mg/kg displayed a lower performance with respect to the vehicle-treated animals. Therefore, the effects exerted by 3 mg/kg L-NAME on rats' spatial memory and the ability of L-NAME to antagonize ketamineinduced performance deficits in the radial water maze were subsequently assessed in two separate studies.

Experiment 3: effects of L-NAME (3 mg/kg) on animals' performance in the radial water maze task

Rats were randomly divided into two experimental groups (10 rats per group) as follows:

vehicle; and L-NAME 3 mg/kg. L-NAME and vehicle were injected every day 60 min before starting testing.

Experiment 4: effects of L-NAME in antagonizing ketamine-induced performance deficits in the radial water maze task

Animals were randomly divided into six experimental groups (10 rats per group) as follows: vehicle+vehicle; vehicle+L-NAME 1 mg/kg; vehicle+L-NAME 10 mg/kg; ketamine 15 mg/kg+vehicle; ketamine 15 mg/kg+L-NAME 1 mg/kg; ketamine 15 mg/kg+L-NAME 10 mg/kg. Control rats were treated with the vehicle 60 and 40 min, respectively, before starting the daily testing. Animals were treated with L-NAME and vehicle 60 and 40 min, respectively before starting testing. Rats received vehicle and ketamine 60 and 40 min, respectively, before starting the daily testing. Rats received L-NAME and ketamine 60 and 40 min, respectively, before starting the daily session. Ketamine administration (10-30 mg/kg) is associated with behavioral symptoms such as hypermotilty and stereotypy which reach the peak effect 15 min after treatment [START_REF] Wesierska | Differential effects of ketamine on the reference and working memory versions of the Morris water maze task[END_REF][START_REF] Verma | NMDA receptor antagonists impair prefrontal cortex function as assessed via spatial delayed alternation performance in rats: modulation by dopamine[END_REF].

Based on these evidences, the daily administration of ketamine was performed 40 min before testing since at this condition ketamine did not induce adverse effects.

Statistical analysis

Data are expressed as mean±SEM. In experiments 1 and 2, results were analyzed using the two-way analysis of variance (ANOVA). Post-hoc comparisons were made by the Tukey's ttest. In experiment 3, reference and working memory incorrect errors were evaluated by the two-way ANOVA with a split-plot design (between-within subjects). Post-hoc comparisons were made by the Tukey's t-test. Spatial probe trial data are calculated as median and interquartile ranges. When comparing among groups the percentage of time spent in the previously reinforced arm, the non-parametric test of Wilcoxon was used.

In experiment 4, the effects of drugs on daily performance (reference and working memory) were analyzed by the three-way ANOVA (two between, one within subjects). Significant interactions between ketamine, L-NAME and trials were further analyzed by comparing ketamine-and L-NAME-treated and non-treated rats at each level separately for the 4 days periods, using the two-way ANOVA with a split-plot design. Post-hoc comparisons were made by the Tukey's t-test. Spatial probe data were analyzed by the non-parametric test of Kruskall-Wallis followed by the Newman-Keuls post-hoc test. A p value <0.05 was considered significant [START_REF] Kirk | Experimental design: procedures for the behavioral science[END_REF].

RESULTS

Experiment 1: effects of L-NAME in antagonizing MK-801-induced performance deficits in the novel object recognition task

A two-way ANOVA analysis of motor activity results demonstrated a main effect either of MK-801 (F(1,72)=18.4, p<0.01) or of L-NAME (F(3,72)=2.93, p<0.05) indicating that rats that received both these compounds displayed higher motility levels as compared to the other groups (Table 1). Total exploration levels were not different among the various experimental groups (Table 1).

Data for index D (Fig. 1) revealed a significant main effect of MK-801 (F(1,72)=24.2, p<0.01), of L-NAME (F(3,72)=4.8, p<0.01) and a significant MK-801 x L-NAME interaction (F(3,72)=3.8, p=0.014). Post-hoc comparisons have shown that the vehicle+MK-801 and the MK-801+L-NAME 10 mg/kg-treated animals were unable to discriminate between the familiar and the novel object as compared to the rest of groups (p<0.05).

Experiment 2: effects of L-NAME in antagonizing ketamine-induced performance deficits in the novel object recognition task

A two-way ANOVA analysis of motility results showed only a significant main effect of ketamine (F(1,72)=29.8, p<0.01) suggesting that animals treated with ketamine exhibited higher locomotor activity levels than the other experimental groups (Table 2). Total exploration levels were not different among the various experimental groups (Table 2).

Concerning D data, a significant main effect of ketamine (F(1,72)=15.8, p<0.01), of L-NAME (F(3,72)=3.4, p<0.05) and a significant interaction between ketamine and L-NAME (F(3,72)=3, p<0.05) was evidenced. Post-hoc analysis has shown that the vehicle+ketamine and the ketamine+L-NAME 10 mg/kg-treated animals were unable to discriminate between the familiar and the novel object as compared with the other groups (p<0.05, Fig. 2).

Experiment 3: effects of L-NAME (3 mg/kg) on animals' performance in the radial water maze task

Reference memory

Data are presented in Fig. 3A. An overall ANOVA showed that both the vehicle and the L-NAME-treated animals made progressively fewer reference memory errors over days (F(3,54)=5.47, p<0.01). In addition, the L-NAME 3 mg/kg-treated animals committed significantly more reference memory errors than their vehicle-treated cohorts (F(1,18)=8.48, p<0.01).

Working memory

Results are illustrated in Fig. 3B. Statistical analysis of the working memory data evidenced that either the vehicle or the L-NAME-treated animals made progressively fewer working memory incorrect errors over days (F(3,54)=9.87, p<0.01). Moreover, the L-NAME 3 mg/kgtreated rats made a significantly higher number of working memory incorrect errors than their vehicle-treated cohorts (F(1,18)=4.4, p=0.05).

Spatial probe trial

Probe analysis comparing percentage of time spent in the previously reinforced quadrant showed an effect of treatment. The 3 mg/kg L-NAME group spent significantly less time in the previously baited channel than the control group (p<0.05, Fig. 3C).

Experiment 4: effects of L-NAME in antagonizing ketamine-induced performance deficits in the radial water maze task

Reference memory

Data are presented in Fig. 4A. Overall ANOVA did not show a significant three-way ketamine x L-NAME x days interaction, neither a significant two-way interaction between ketamine and L-NAME, between ketamine and days and between L-NAME and days. A significant main effect of ketamine (F(1,216)=25.1, p<0.01), of days (F(3,216)=11.1, p<0.01) but not of L-NAME however was revealed.

Additional analyses of rats' performance in this spatial reference memory task were carried out by split-plot ANOVAs. The ketamine+vehicle-treated animals committed significantly more reference memory errors than their vehicle+vehicle-treated cohorts (F(1,18)=40.6, p<0.01). L-NAME, at any dose, failed to counteract this ketamine-induced reference memory impairment.

Animals treated with 1 mg/kg L-NAME+ketamine made more reference memory errors than the vehicle+L-NAME 1 mg/kg-treated rats (F(1,18)=7.3, p=0.014). Choice accuracy abilities, in terms of number of reference memory errors, of the 10 mg/kg L-NAME+ketamine-treated rats and those animals that received vehicle+L-NAME 10 mg/kg were not different.

Working memory

Results are presented in Fig. 4B. Overall ANOVA did not show a significant three-way ketamine x L-NAME x days interaction, neither a significant two-way interaction between ketamine and days and between L-NAME and days. Interestingly, a significant two-way interaction between ketamine and L-NAME was evidenced (F(2,216)=3.64, p<0.05) indicating that the rates of learning were different among the various groups. In addition, a significant main effect of ketamine (F(1,216)=28.1, p<0.01), of days (F(3,216)=8, p<0.01) but not of L-NAME was evidenced.

Further analyses of animals' performance in this spatial working memory paradigm were carried out by split-plot ANOVAs. Animals that received ketamine+vehicle commtted significantly more working memory incorrect errors than their vehicle+vehicle counterparts (F(1,18)=17.9, p<0.01). Rats that received 10 mg/kg L-NAME+ketamine made significantly fewer working memory incorrect errors than their counterparts treated with ketamine+vehicle (F(1,18)=8.35, p<0.01). In addition, these rats performance, in terms of working memory incorrect errors, was not different than that displayed by their vehicle cohorts (10 mg/kg L-NAME+vehicle-treated rats). Animals treated with 1 mg/kg L-NAME+ketamine made a higher number of working memory incorrect errors than the vehicle+L-NAME 1 mg/kgtreated rats (F(1,18)=13.8, p<0.01).

Spatial probe trial

Probe analysis comparing percentage of time spent in the previously reinforced quadrant showed an effect of treatment (H=25.8, p<0.01, Fig. 4C). Ketamine+vehicle and ketamine+L-NAME 1 mg/kg-treated animals spent less time in the previously baited channel as compared to the respective control populations (Newman-Keuls, post-hoc test; p<0.05). L-NAME, at 10 mg/kg, attenuated this ketamine-induced impairment (p<0.05 vs ketamine+vehicle-treated animals).

DISCUSSION

The major findings of the present study have shown that the NMDA receptor antagonists MK-801 and ketamine induced performance deficits in different cognitive tasks in the rat.

These cognitive impairments could be antagonized by the NOS inhibitor L-NAME.

Novel object recognition task

Our results are in line with previous studies in which post-training administration of both MK-801 and ketamine disrupted rats' performance in the novel object recognition task [START_REF] Pitsikas | Nitric oxide donor molsidomine attenuates psychotomimetic effects of the NMDA receptor antagonist MK-801[END_REF][START_REF] Pitsikas | Effects of sub-anesthetic doses of ketamine on rats' spatial and non-spatial recognition memory[END_REF]. A single post-training injection of L-NAME (1 and 3, but not 10 mg/kg) attenuated either the MK-801 or the ketamine-induced performance deficits in this recognition memory paradigm. MK-801, ketamine and L-NAME influenced rats' performance during retention, seemingly reflecting a modulation of post-training mnemonic processes (storage and/or retrieval of information).

Compounds were administered systemically, thus, it cannot be excluded that non-specific factors (attentional, sensorimotor deficits) might have influenced animals' performance. All animals that received these two NMDA receptor antagonists displayed higher levels of motility, but not of general exploration during T2, with respect to their control cohorts.

Treatment with L-NAME did not antagonize this NMDA blockade-induced hyperactivity but successfully counteracted recognition memory deficits. This pattern of results implies that the effects of the compounds on rats' cognitive performance were unrelated to the extent of motility and exploratory behavior.

Present results are in agreement with our previous findings showing that L-NAME did not impair recognition memory but it reversed delay-dependent impairments in the novel object recognition task, especially at lower doses [START_REF] Boultadakis | Effects of the nitric oxide synthase inhibitor L-NAME on different memory components as assessed in the object recognition task in the rat[END_REF].

Radial water maze task

Data have shown that L-NAME (1 and 10 mg/kg) alone did not disrupt animals' spatial learning abilities. A per se effect of L-NAME was observed when this NOS inhibitor was delivered at the dose of 3 mg/kg, however. Those animals' spatial learning abilities were poorer as compared to those displayed by the vehicle+vehicle-treated subjects. Rats that received ketamine performed less efficiently than the vehicle+vehicle and the L-NAME (1 and 10 mg/kg)+vehicle-treated rats suggesting that this NMDA receptor antagonist disrupted both spatial reference and spatial working memory. The higher dose of L-NAME (10 mg/kg) attenuated ketamine-induced working, but not reference, memory deficits. Both ketamine+L-NAME groups expressed a lower performance as compared to the respective cohorts treated with L-NAME+vehicle.

When the ability of rats to use the extramaze cues surrounding the radial water maze was evaluated (spatial probe trial) all control rats persistently swam more in the previously reinforced arm of the maze, while L-NAME 3 mg/kg+vehicle, ketamine+vehicle and ketamine+L-NAME 1 mg/kg-treated animals were unable to do so. L-NAME (10 mg/kg) attenuated this ketamine produced disrupting effect.

The radial water maze test evaluates rodents' spatial memory. The procedure employed in the present study offers the possibility to assess in the same animal simultaneously two different aspects of memory (reference and working) [START_REF] Jarrard | On the role of the hippocampal connections in the performance of place and cue tasks: comparisons with damage to hippocampus[END_REF][START_REF] Pitsikas | Deterioration of spatial and nonspatial reference and working memory in aged rats: protective effect of life-long calorie restriction[END_REF]. This within-subject and within-task design is advantageous because each dissociation is obtained from the same animal at the same time, minimizing many sources of variability such as task magnitude of reinforcement, motivational, perceptual and motor processes [START_REF] Olton | Memory functions and hippocampus[END_REF].

Our results of the radial water maze paradigm are consistent with previous reports in which the effects of L-NAME (10 mg/kg) on glutamatergic hypofunction were assessed (Wass et al, 2006a;Wass et al, 2006b). The outcome of these studies was that treatment with this NOS inhibitor antagonized PCP-induced spatial reference, spatial working memory, but not retention deficits in a modified Morris water maze procedure (Wass et al, 2006a;Wass et al, 2006b).

A potential issue when using NOS inhibitors relates to its hypertensive properties. It is difficult, therefore, to quantify how and at what extent these cardiovascular effects might have specifically affected animals' cognitive performance. Reportedly, NOS inhibitors injected peripherally induce a nearly maximal hypertensive effect at 10 mg/kg [START_REF] Rees | Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and vivo[END_REF].

Under our experimental conditions, L-NAME, administered systemically at 10 mg/kg, attenuated ketamine-induced performance deficits in the radial water maze task. This implies that the effects of this compound on rats' cognitive performance were unrelated to its potential hypertensive action.

NMDA hypofunction and nitric oxide

Pharmacological blockade of NMDA receptors has been proposed as a relevant model of schizophrenia in humans and animals. [START_REF] Javitt | Recent advances in the phencyclidine model of schizophrenia[END_REF][START_REF] Geyer | Animal models of psychiatric disorders[END_REF][START_REF] Krystal | Sub-anesthetic effects of the noncompetitive NMDA antagonist, ketamine, in humans. Psychotomimetic, perceptual, cognitive and neuroendocrine responses[END_REF]. The NMDA antagonist treatment model has excellent face validity for cognitive symptoms of schizophrenia because an exposure to these drugs impairs PFCdependent cognitive functions in a manner that is similar to schizophrenia [START_REF] Javitt | Recent advances in the phencyclidine model of schizophrenia[END_REF][START_REF] Krystal | Sub-anesthetic effects of the noncompetitive NMDA antagonist, ketamine, in humans. Psychotomimetic, perceptual, cognitive and neuroendocrine responses[END_REF]. PCP, ketamine and MK-801 produce their psychotomimetic effects by blocking NMDA receptors located on GABA interneurons, resulting in decreased firing of GABAergic inhibitory neurons and, thereby, increased excitability in limbic circuits [START_REF] Olney | NMDA antagonist neurotoxicity: mechanism and prevention[END_REF][START_REF] Moghaddam | Activation of glutamatergic neurotransmission by ketamine: a novel step in the pathway from NMDA receptor blockade to dopaminergic and cognitive disruptions associated with the prefrontal cortex[END_REF]. This disinhibitory action elicits an increase in terms of neuronal activity and excessive glutamate and dopamine release in the PFC and limbic regions [START_REF] Moghaddam | Activation of glutamatergic neurotransmission by ketamine: a novel step in the pathway from NMDA receptor blockade to dopaminergic and cognitive disruptions associated with the prefrontal cortex[END_REF][START_REF] Lorrain | Effects of ketamine and N-methyl-D-aspartate on glutamate and dopamine release in the rat prefrontal cortex: modulation by a group II selective metabotropic glutamate receptor agonist LY379268[END_REF][START_REF] Razoux | Ketamine at a dose that disrupts motor behavior and latent inhibition, enhances prefrontal cortex synaptic efficacy and glutamate release in the nucleus accumbens[END_REF].

NOS inhibitors may exert both excitatory and inhibitory influences on NMDA receptor associated events. NO's dual action as mediator and negative modulator of glutamate neurotransmission at the NMDA receptor complex give this system a unique role in regulating the balance of excitatory and inhibitory influences. It is this balance of excitatory and inhibitory processes that is likely to be disrupted in schizophrenia, as proposed by the glutamatergic hypofunction [START_REF] Dawson | Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures[END_REF].

Our findings indicate that L-NAME, for the first time to our knowledge, antagonized both MK-801 and ketamine-induced recognition memory deficits suggesting that this NOS inhibitor is also effective against NMDA blockade produced by the action of NMDA antagonists other than PCP. Furthermore, L-NAME was found to attenuate spatial memory deficits produced by ketamine in the rat. The latter, support and extend previous results in which spatial memory deficits induced by another NMDA antagonist (PCP) were counteracted by this NOS inhibitor (Wass et al, 2006a;Wass et al, 2006b). Collectively, the present results further suggest an involvement of NO in the NMDA hypofunction-induced cognitive deficits.

Contrary to the expectations, when L-NAME was administered alone at 3 mg/kg, impaired rats' spatial but not recognition memory abilities. It remains uncertain if this dual effect of L-NAME on memory was related to the task specificity, the type of memory studied and/or the different pharmacological design utilized.

The radial water maze, is a negatively reinforced paradigm assessing spatial memory. The novel object recognition test, is a task that does not involve at all, the learning of a rule since it is based on the spontaneous exploratory behavior of rodents evaluating recognition memory [START_REF] Ennaceur | One-trial object recognition in rats and mice: Methodological and theoretical issues[END_REF]. In the novel object recognition task rats received a single injection of compounds just after the "sample" trial T1. Conversely, in the radial water maze paradigm, treatment was applied once per day 60 min before starting the daily testing, for 5 consecutive days.

Interestingly, this impairing effect of L-NAME on spatial memory, was not seen at the "side" doses of 1 and 10 mg/kg revealing thus, an U-shaped dose-effect curve. At present, the biological bases of U dose-response relationship are unknown, although receptor fatigue or tachyphylaxis [START_REF] Day | Autonomic Pharmacology. Experimental and Clinical aspects[END_REF] has been suggested as potential mechanisms [START_REF] Martinez | Memory: drugs and hormones[END_REF].

Moreover, L-NAME has also been reported to act as a partial agonist in the NO system, antagonizing or stimulating NO synthesis in different tissues [START_REF] Archer | NG-mono-methyl-L-arginine causes nitric oxide synthesis in isolated arterial rings: trouble in paradise[END_REF].

Further studies are needed however, in order to clarify this important issue. The mechanism(s) of action of low doses of NOS inhibitors on learning and memory is not yet clarified and is matter of investigation. It has been demonstrated that the neuroprotective effects of NOS inhibitors were obtained when inhibition of NO production was only mild and transient. Conversely, long-lasting inhibition of NO production by high doses of NOS inhibitors leads to neurotoxicity [START_REF] Contestabile | Brain nitric oxide and its dual effect in neuroprotection/neurodegeneration: understanding molecular mechanisms to devise drug approaches[END_REF]. Small changes in local NO concentration and the time of administration therefore, may be a key factor in determining its biological action [START_REF] Contestabile | Brain nitric oxide and its dual effect in neuroprotection/neurodegeneration: understanding molecular mechanisms to devise drug approaches[END_REF].

It has previously been reported that a wide range of translational PCP-induced effects including deficits in preattentive information processing, nonassociative learning, selective attention, spatial memory can all be prevented by interfering with the production of NO [START_REF] Johansson | Nitric oxide synthase inhibition blocks phencyclidine-induced behavioural effects on prepulse inhibition and locomotor activity in the rat[END_REF][START_REF] Klamer | The nitric oxide synthase inhibitor, L-NAME, block phencyclidine-induced disruption of prepulse inhibition in mice[END_REF][START_REF] Klamer | The neuronal selective nitric oxide synthase inhibitor, Nomega-propyl-L-arginine, blocks the effects of phencyclidine on prepulse inhibition and locomotor activity in mice[END_REF]Klamer et al, 2005a;Klamer et al, 2005b). These findings suggest that the schizophrenia-like behavioral effects of PCP in rodents are, at least in part, mediated by an increase in NO activity [START_REF] Palsson | Increased cortical nitric oxide release after phencyclidine administration[END_REF]. This is supported by a recent study showing a NO-dependent increase in cGMP signaling, a main effector of NO in the brain, in the mouse PFC following PCP administration [START_REF] Fejgin | Nitric oxide signaling in the medial prefrontal cortex is involved in the biochemical and behavioral effects of phencyclidine[END_REF]. In this context, it has been found that L-NAME attenuated sensorimotor gating deficits

Legend to figures

Fig. 1 .

 1 Fig. 1. Novel object recognition task. Vehicle, MK-801 and L-NAME were injected i.p., just

Fig. 2 .

 2 Fig. 2. Novel object recognition task. Vehicle, ketamine and L-NAME were injected i.p., just

Fig. 3 .

 3 Fig. 3. Radial water maze task. Vehicle and L-NAME were injected i.p., every day 60 min

Fig. 4 .

 4 Fig. 4. Radial water maze task. Vehicle, ketamine and L-NAME were injected i.p., every day

Table 1 .

 1 Effects of MK-801 and L-NAME on rats' performance in the novel object recognition task N=number of rats. Compounds were injected intraperitoneally immediately after T1.

			T2	T2
	Group	N	Motor activity (number of steps)	Exploration time (s.)
			Mean±SEM	Mean±SEM
	Vehicle+vehicle	10	47.5±1.8	12.5±0.3
	Vehicle+L-NAME (1 mg/kg)	10	41.8±5.2	12.2±0.4
	Vehicle+L-NAME (3 mg/kg)	10	35.5±2.1	11.6±1.3
	Vehicle+L-NAME (10 mg/kg)	10	44.4±3.9	12.5±0.6
	MK-801 (0.1 mg/kg)+vehicle	10	58.6±3.5	11.9±0.5
	MK-801+L-NAME (1 mg/kg)	10	55.4±3.7	14.2±0.8
	MK-801+L-NAME (3 mg/kg) MK-801+L-NAME (10 mg/kg)	10 10	47.2±5.1 54.1±3.6	12.4±0.9 12.4±0.4

Table 2 .

 2 Effects of ketamine and L-NAME on rats' performance in the novel object recognition task N=number of rats. Compounds were injected intraperitoneally immediately after T1.

			T2	T2
	Group	N	Motor activity (number of steps)	Exploration time (s.)
			Mean±SEM	Mean±SEM
	Vehicle+vehicle	10	45.4±2.6	12.7±0.7
	Vehicle+L-NAME (1 mg/kg)	10	46.9±4.5	14.2±1
	Vehicle+L-NAME (3 mg/kg)	10	46.4±5.2	13.6±0.7
	Vehicle+L-NAME (10 mg/kg)	10	45.1±4.9	14.1±1.3
	Ketamine (3 mg/kg)+vehicle	10	67.6±1.7	14.5±0.8
	Ketamine+L-NAME (1 mg/kg)	10	57.8±3.3	13.3±0.5
	Ketamine+L-NAME (3 mg/kg) Ketamine+L-NAME (10 mg/kg)	10 10	58.7±3.3 59.1±4.3	12.7±0.8 13.2±0.9

The present study was supported by a grant of the Research Committee of the University of Thessaly (no. 3689) to N.P.

caused by PCP, by reducing PCP-induced increase in cyclic GMP (cGMP) production in the PFC of the mouse brain [START_REF] Fejgin | Nitric oxide signaling in the medial prefrontal cortex is involved in the biochemical and behavioral effects of phencyclidine[END_REF]. This mechanism might be a plausible explanation for the beneficial effects of low doses of L-NAME on memory impairments related to glutamatergic hypofunction. Further studies however, are required in order to elucidate this important issue.

Concluding remarks

In summary, studies here-in presented demonstrate that L-NAME was capable in antagonizing MK-801 and ketamine-induced memory deficits and this beneficial action exerted by L-NAME on cognition was observed at a different dose range. This, in turn, indicates that L-NAME is sensitive to glutamatergic hypofunction produced by other than PCP NMDA receptor antagonists such as MK-801 and ketamine. In addition, the present results show that L-NAME alone differentially affects rodents' spatial memory. The latter suggest a careful consideration of the balance between neurodegenerative and neuroprotective effects produced by this NOS inhibitor since L-NAME might constitute a potential candidate for the treatment of schizophrenia.

DISCLOSURE/CONFLICT OF INTEREST

The authors hereby declare that no financial support or compensation has been received from any individual or corporate entity over the past 3 years for research or professional service and there are no personal financial holdings that could be perceived as constituting a potential conflict of interest.

ACKNOWLEDGEMENTS