Pre-aggregated Aβ25–35 alters arginine metabolism in the rat hippocampus and prefrontal cortex

Neuroscience 193 (2011) 269 –282

PRE-AGGREGATED A␤25–35 ALTERS ARGININE METABOLISM IN THE RAT HIPPOCAMPUS AND PREFRONTAL CORTEX PING LIU,a,c* YU JING,a,c NICOLA D. COLLIE,a,c SASKIA A. CAMPBELLc AND HU ZHANGb,c

ence of extracellular senile plaques (SPs) and intracellular neurofibirillary tangles (NFTs) in the brain (Hyman et al., 1989; Van Hoesen et al., 2000). Amyloid beta (A␤) is a 39to 43-amino acid peptide derived from proteolytic cleavage of amyloid precursor protein, and increased levels of pathogenic A␤ peptides have been implicated in the etiology of AD (Hardy and Higgins, 1992; Selkoe, 2001). The 11-amino acid peptide A␤25–35 is the neurotoxic core of the full-length A␤ and is even more toxic than parent peptide (Varadarajan et al., 2000, 2001). There is evidence suggesting that A␤25–35 may be related to AD pathogenicity (for a review see Kaminsky et al., 2010). It has been reported, for example, that A␤25–35 is produced in brains of AD patients by enzymatic cleavage of the naturally occurring A␤1– 40, and the titer of serum antibodies against aggregates of A␤25–35 is increased in patients with progressive AD (Kubo et al., 2002; Gruden et al., 2004). A␤25–35 has been used as an experimental tool to understand the toxicity of A␤ and its contributions to cognitive deficits in AD. Oligomeric A␤25–35 activates astrocytes and microglial cells, alters the function of cerebrovascular endothelial cells, causes perturbation of Ca2⫹ homeostasis, protein oxidation, lipid peroxidation, neuronal apoptosis and death, and impairs synaptic plasticity (Blanchard et al., 1997; Sun and Alkon, 2002; Stepanichev et al., 2004; Yin et al., 2005; Bisel et al., 2007; Klementiev et al., 2007; Resende et al., 2007; Alkam et al., 2008; Chisari et al., 2010). In rodents, a single unilateral or bilateral i.c.v. infusion/injection of pre-aggregated A␤25–35 results in learning and memory impairments at various time points after the administration of A␤ (Maurice et al., 1996; Delobette et al., 1997; Yamaguchi and Kawashima, 2001; Sun and Alkon, 2002; Stepanichev et al., 2003, 2004, 2005, 2006; Cheng et al., 2006; Klementiev et al., 2007; Bergin and Liu, 2010; for a review see Gulyaeva and Stepanichev, 2010). A growing body of evidence suggests that arginine, a semi-essential amino acid, and its metabolites play a prominent role in AD pathogenesis (for reviews see Law et al., 2001; Malinski, 2007; Yi et al., 2009). Arginine is metabolized by nitric oxide synthase (NOS) to produce nitric oxide (NO) and citrulline (Wu and Morris, 1998). There are three isoforms of NOS—neuronal NOS (nNOS) and endothelial NOS (eNOS) are Ca2⫹/calmodulin-dependent constitutive forms, whereas inducible NOS (iNOS) is Ca2⫹/ calmodulin-independent and is usually only produced in response to pathological stimuli (Zhang and Snyder, 1995). Although NO serves as a neural messenger and regulates cerebral blood flow at the physiological level, excessive amounts of NO can react with superoxide to

a Department of Anatomy and Structural Biology, University of Otago, Dunedin, New Zealand b

School of Pharmacy, University of Otago, Dunedin, New Zealand

c

Brain Health Research Centre, University of Otago, Dunedin, New Zealand

Abstract—Amyloid beta (A␤) has been proposed to play a central and causative role in the development of Alzheimer’s disease. A␤25–35, the neurotoxic domain of the full-length A␤, causes learning and memory impairments in rodents. The present study investigated the effects of a single bilateral i.c.v. infusion of pre-aggregated A␤25–35 (30 nmol/rat) on animals’ performance in the open field, and on arginine metabolic enzymes and metabolites in the CA1, CA2/3, and dentate gyrus (DG) sub-regions of the hippocampus and prefrontal cortex (PFC) at the time point of 6 – 8 days after A␤ infusion. A␤25–35 rats displayed reduced exploratory activity in the open field relative to the A␤35–25 (reverse peptide; 30 nmol) rats. A␤25–35 resulted in significantly decreased nitric oxide synthase (NOS) activity and endothelial NOS protein expression, but increased arginase activity, arginase II protein expression, and ornithine and putrescine levels, in hippocampal CA2/3. There were increased glutamate and putrescine levels in the DG, but decreased agmatine levels in the DG and PFC, in the A␤25–35 group relative to the A␤35–25 one. Cluster analyses were performed to determine if the nine related neurochemical variables (arginine, citrulline, ornithine, agmatine, putrescine, spermidine, spemine, glutamate, and GABA) formed distinct groups, and whether it changed as a function of A␤25–35. There were substantially different clusters between the two groups in the hippocampus and PFC. These results demonstrate that A␤25–35 alters arginine metabolism, which further supports the prominent role of arginine and its metabolites in Alzheimer’s disease (AD) pathogenesis. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: amyloid beta, arginine, nitric oxide synthase, polyamines, agmatine, hippocampus.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive memory loss and the pres*Corresponding author. Tel: 64-03-4797536; Fax: ⫹64-03-4797254. E-mail address: [email protected] (P. Liu). Abbreviations: A␤, amyloid beta; AD, Alzheimer’s disease; ADC, arginine decarboxylase; ANOVA, analysis of variance; DG, dentate gyrus; EGTA, ethylene glycol tetraacetic acid; eNOS, endothelial nitric oxide synthase; HPLC, high performance liquid chromatography; iNOS, inducible nitric oxide synthase; LC/MS, liquid chromatography/mass spectrometry; NFTs, neurofibirillary tangles; NMDA, N-methyl-D-aspartate; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NOS, nitric oxide synthase; ODC, ornithine decarboxylase; PFC, prefrontal cortex; SPs, senile plaques.

0306-4522/11 $ - see front matter © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2011.07.054

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generate the potent oxidant peroxinitrite (ONOO⫺), which induces nitroxidative stress, apoptosis, and an inflammation cascade; breaks DNA strands; and results in mitochondrial dysfunction (Law et al., 2001; Malinski, 2007). It has been shown that A␤ stimulates NO production, promotes or exacerbates local inflammation, induces endothelial apoptosis and dysfunction, and triggers mitochondrial fission, synaptic loss, and neuronal damage (Dickson et al., 1993; El Khoury et al., 1996; Tran et al., 2001; Yin et al., 2005; Alkam et al., 2008; Cho et al., 2009; Limón et al., 2009a,b; Chisari et al., 2010). Arginase metabolizes arginine to ornithine and urea (Wu and Morris, 1998; Wiesinger, 2001). There are two isoforms of arginase in mammals: arginase I is a cytosolic enzyme that is highly expressed in liver, whereas arginase II, a mitochondrial enzyme, is expressed at low levels in kidney, brain, and small intestine, with little or no expression in the liver. There is evidence suggesting the presence of both arginase I and II in mammalian brains (Braissant et al., 1999; Yu et al., 2001; Colton et al., 2006; Hansmannel et al., 2010). Polyamines putrescine, spermidine, and spermine are downstream metabolites of arginine. In mammalian cells, de novo synthesis from ornithine by ornithine decarboxylase (ODC) appears to be the major route to the production of polyamines, and the physiological concentrations of polyamines are essential for cells to grow and to function in an optimal manner (for reviews see Williams, 1997; Wallace, 2000; Oredsson, 2003; Wallace et al., 2003). In the AD brains, there are elevated ODC protein levels and altered sub-cellular localization of ODC and polyamine content (Morrison and Kish, 1995; Morrison et al., 1995, 1998; Nilsson et al., 2006). It has been reported that A␤25–35 increased spermidine uptake and elevated ODC activity, and that A␤1– 42 resulted in increased putrescine, sperminine, and spermine levels in cultured neurons (Yatin et al., 1999, 2001). These findings suggest altered polyamine metabolism in AD (see Yi et al., 2009 for a review). Arginine can also be metabolized by arginine decarboxylase (ADC) to form agmatine and carbon dioxide (Wu and Morris, 1998). Li et al. (1994) first reported the presence of agmatine and ADC in mammalian brains. Agmatine is a putative novel neurotransmitter, interacts with a number of receptor subtypes, and regulates the production of NO and polyamines as an endogenous regulator (for reviews see Reis and Regunathan, 2000; Satriano, 2003; Halaris and Plietz, 2007). Since agmatine is metabolized by agmatinase to form putrescine, it also serves as a precursor of polyamines and is therefore considered to be a member of the polyamine family (Moinard et al., 2005). Recent evidence suggests that endogenous agmatine may directly participate in the processes of learning and memory as a novel neurotransmitter (Liu et al., 2008a, 2009a; Leitch et al., 2011), and that exogenous agmatine has anti-inflammatory and neuroprotective effects (Regunathan et al., 1999; Feng and LeBlanc, 2003; Arndt et al., 2009; Hong et al., 2009). Abe et al. (2000) reported that agmatine effectively suppressed the production of NO induced by interferon-␥ and A␤1– 40. Rushaidhi et al. (2008)

demonstrated that agmatine significantly suppressed agerelated elevation of NOS activity in the hippocampus and prefrontal cortex. Bergin and Liu (2010) investigated the effects of a single bilateral i.c.v. infusion of pre-aggregated A␤25–35 (30 nmol) in a battery of behavioral tests conducted during the period 4 –14 weeks after A␤25–35 infusion, and evaluated the protective effect of agmatine (40 mg/kg, i.p.) administered 30 min prior to A␤25–35 infusion and once daily for a further nine consecutive days. Agmatine treatment significantly protected against A␤25–35-induced learning and memory deficits in the water maze, the object recognition memory task, and the radial arm maze task. These findings suggest that the first 9 days after A␤25–35 infusion may be a critical treatment window for agmatine. However, it is unclear how pre-aggregated A␤25–35 affects endogenous agmatine and other metabolic pathways of arginine during this period. The present study was designed to investigate the effects of A␤25–35 on animals’ behavior (as an indication of the toxicity) and the three metabolic pathways of arginine at the time points of 6 – 8 days post-infusion in a single study. Animals’ general behavior was assessed in the open field on day 6 following a single bilateral infusion of pre-aggregated A␤25–35 (30 nmol/rat) or A␤35–25 (the reverse peptide; 30 nmol/rat). Animals were sacrificed on day 8 post-infusion, and the hippocampus and the prefrontal cortex were harvested to measure the activities and protein levels of NOS and arginase, and the tissue concentrations of arginine and its metabolites (citrulline, ornithine, agmatine, putrescine, spermidine, and spermine). Glutamate and GABA are the major excitatory and inhibitory neurotransmitters in the central nervous system, respectively. Since ornithine can be converted to L-glutamylc-semialdehyde that is further metabolized to glutamate by P5C dehydrogenase (Wu and Morris, 1998), we also measured the tissue concentrations of glutamate and GABA in the hippocampus and the prefrontal cortex. Because there is a functional dissociation across the CA1, CA3, and dentate gyrus (DG) (for a review see Kesner et al., 2004), A␤25–35-induced neurochemical changes in the hippocampus was examined at the sub-regional level.

EXPERIMENTAL PROCEDURES Subjects Sixteen male Sprague–Dawley rats, weighing between 330 and 400 g, were housed one per cage (33⫻21.5⫻17.5 cm3) with free access to food and water, and maintained on a 12-h light/dark cycle (lights on at 8 AM). Surgical and behavioral procedures were conducted during the light period of the light– dark cycle. All experimental procedures were carried out in accordance with the regulations of the University of Otago Committee on Ethics in the Care and Use of Laboratory Animals. Every attempt was made to limit the number of animals used and to minimize their suffering.

Surgery Rats were anesthetized by halothane and placed in a stereotaxic apparatus. Lopaine (3 mg/kg) was injected subcutaneously at the surgical site before a midline incision was made and the scalp retracted to expose the skull. Holes were drilled bilaterally into the

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Behavioral procedures

Fig. 1. Experimental timeline. Animals received a single bilateral intracerebroventricular (i.c.v.) infusion of pre-aggregated A␤35–25 or A␤25–35 at a dose of 30 nmol/rat. All animals were then tested in the open field on day 6, and sacrificed on day 8 after A␤ infusion.

skull at 0.6 mm posterior to bregma and 1.4 mm lateral to the midline. A 23-gauge cannula, which was connected to a 10-␮l Hamilton syringe by plastic tubing, was inserted into each lateral ventricle at a depth of 3.8 mm from the dura. The surgical coordinates were derived from Paxinos and Watson (1998) and based on our previous studies (Liu et al., 2008d; Liu and Bergin, 2009; Bergin and Liu, 2010). A␤35–25 or A␤25–35 was infused into each lateral ventricle over 2 min, and the cannula was allowed to remain in the ventricle for at least 2 min to allow diffusion of the peptide. Successful infusion was confirmed by air bubble movement in the plastic tubing. The wound was then sutured using 9-mm stainless steel wound clips (Reflex, CellPoint Scientific Inc, USA). Following surgery, each rat was kept warm and monitored until spontaneous movement occurred. Strepcin (0.1 ml) was used s.c. for post-operative infection control and Carprofen (5 mg/kg s.c.) was used for post-operative analgesia. Animals’ wound, body weight, water intake, and general behavior were closely monitored for at least 5 days post-surgery. The behavioral test was started on day 6 after A␤ infusion (Fig. 1).

Drug and treatment The rats were randomly allocated to the A␤35–25 (n⫽8) or A␤25–35 (n⫽8) group. The variation in animals’ body weights was considered and counterbalanced across the two groups. A␤25–35 and the reverse peptide A␤35–25 were purchased from Bachem (Australia). Both peptides were dissolved in sterile bidistilled water at a concentration of 3 nmol/␮l, and were “aged” before surgery by incubation at 37 °C for 4 days (Maurice et al., 1996; Bergin and Liu, 2010). Light microscopic observation demonstrated the existence of insoluble fibrillar like assemblies in the A␤25–35, but not A␤35–25, solution as reported previously (Bergin and Liu, 2010). A␤35–25 (15 nmol/5 ␮l) or A␤25–35 (15 nmol/5 ␮l) was infused into each lateral ventricle, and the doses were based on previous studies (Stepanichev et al., 2005; Yenkoyan et al., 2009; Bergin and Liu, 2010).

Behavioral apparatus Animals were tested in a windowless room with three clear and one red 75-W bulbs mounted on the ceiling. A video camera was mounted at ceiling height in the center of the room and used for recording animal’s performance during the experimental period. A radio speaker was located adjacent to the video camera at ceiling height to provide background masking noise. The extramaze cues (the laboratory furniture, lights and several prominent visual features on the walls, as well as the location of the experimenter) were held constant during the test. The open field consisted of a 60⫻60-cm2 wooden box with identical walls 20 cm high. All four of the chamber walls and the floor of the box were painted black, and the floor was divided into 36 equal-sized squares. The box was elevated approximately 60 cm above the floor.

All animals were tested in the open field on day 6 after A␤ infusion (Fig. 1). Animals’ exploratory behavior was tested by placing them into the open-field chamber for a period of 5 min. Animal’s performance was videotaped and analyzed offline by a computerized tracking system (HVS, 2020) (Liu et al., 2008d; Gupta et al., 2009; Liu and Bergin, 2009; Liu and Collie, 2009; Bergin and Liu, 2010). The duration of wall-supported rearings and groomings, the path length animals traveled, the percentage of maze used, the percentage of time moving in the apparatus, and the percentage of time spent in the outer zone (10 cm from the wall), middle zone, and center (central four 10-cm squares) were analyzed.

Sample preparation and histology On day 8 post-A␤ infusion, all rats were sacrificed by decapitation without anesthesia (Fig. 1). The brains were rapidly removed and left in cold saline (4 °C) for at least 45 s. The sub-regions of the hippocampus (CA1, CA2/3, and DG) and the prefrontal cortex (PFC) were dissected freshly on ice (Liu et al., 2004a, 2008a,b,c, 2009a,b). The tissues harvested from one hemisphere were frozen immediately and stored at ⫺80 °C until the NOS and arginase assays and Western blot. The tissues collected from the other hemisphere were then weighed, homogenized in ice-cold 10% perchloric acid (⬃50 mg wet weight per millilitre), and centrifuged at 10,000 rpm for 10 min at 4 °C to precipitate protein. The supernatants (the perchloric acid extracts) were frozen immediately and stored at ⫺80 °C until the high-performance liquid chromatographic (HPLC) and liquid chromatography/mass spectrometric (LC/MS/MS) assays. The anterior portion of each brain was collected after the PFC was dissected out, and immersed in 10% (wt/vol) formalin in 0.9% saline for at least 2 weeks. The brains were then switched to a 30% (w/v) sucrose–formalin solution for 3–5 days. Each brain was sectioned (60 ␮m) in the coronal plane by vibratome, mounted on slides, and stained with Thionin. The cannulae tracks for each brain were then examined.

NOS and arginase assays At the time of the assays, protease-inhibitory buffer containing 50 mM Tris–HCL (pH 7.4), 10 ␮M phenylmethylsulfonyl fluoride, 15 ␮M pepstatin A, and 2 ␮M leupeptin (1:10 w/v) was added to the samples on ice. Brain tissues were then homogenized using ultrasonification (Branson Sonifier 150D, Branson Ultrasonics Corporation, CT, USA) and centrifuged at 12,000 g for 10 min at 4 °C. Protein concentrations in the supernatant were measured based on the Bradford method (Bradford, 1976) using a Bio-Rad protein assay dye reagent concentrate and Bio-Rad Benchmark Plus microplate spectrophotometer (Bio-Rad Laboratories Inc., CA, USA) (Liu et al., 2003a,b, 2004a,b, 2005, 2009b). Each supernatant was then separated into three parts and used for the NOS and arginase assays and Western blot, respectively. To analyze NOS activity, we employed the radioenzymatic assay technique developed by Bredt and Snyder (1990). Total NOS enzyme activity was measured as the ability of tissue homogenates to convert [3H] L-arginine to [3H] L-citrulline in the presence of co-factors as described previously (Vane et al., 1994; Liu et al., 2003a,b, 2004a,b, 2005, 2009b). All assays were performed in duplicate. For each brain region, the tissues from the two groups were processed at the same time and the order was counterbalanced. Samples were incubated at 37 °C for 30 min in the presence of 1 mM nicotinamide adenosine dinucleotide phosphate (NADPH), 30 nM calmodulin, 50 mM L-valine, 5 ␮M tetrahydrobiopterin, 2 mM calcium, and a mixture of unlabeled L-arginine and 10 nM of [3H]arginine (specific radioactivity: 40 Ci/mmol; Amersham, New Zealand.). In experiments to determine the contribution of iNOS (calcium independent) to total NOS activity,

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calcium was replaced with 1 mM ethylene glycol-bis(␤-aminoethyl ether)-N,N,N’N’-tetraacetic acid (EGTA). The reaction was stopped by adding 1 ml of 20 mM n-2-hydroxyethylpiperazine-N=2-ethanesulfonic acid (HEPES) buffer (containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM EGTA, pH 5.5) into the tube and transferring the tube onto ice. The newly formed [3H] 3 L-citrulline was separated from the [ H] L-arginine by passing the mixture over 1 ml Dowex columns (1:1 (v/v) Dowex/H2O-50W (200 – 400, 8% cross-linked, Na⫹ form; Sigma, Sydney, Australia). The eluted labeled material was quantified by liquid scintillation spectroscopy, using a Beckman liquid scintillation counter (LS 3801, USA). The counts-per-minute (cpm) for duplicate samples were averaged and corrected with respect to the blank control and background radioactivity. NOS activity was expressed as pmol [3H] L-citrulline/30 min/mg protein. The measurement of arginase activity was the same as we have previously described (Liu et al., 2003a,b, 2004a,b, 2005, 2009b). For each brain region, the tissues from the two groups were processed at the same time and the order was counterbalanced. In brief, 10 mM MnCl2 in 50 mM Tris–HCL (pH 7.5) was added to each sample and the enzyme activated by heating at 55 °C for 10 min. The substrate, 0.5 M L-arginine, was then added and incubated at 37 °C for 1 h, after which the reaction was terminated by the addition of an acid mix: H2SO4:H3PO4:H2O (1:3:7). The urea formed was then assessed spectrophotometrically at 540 nm after the addition of 9% 1-phenyl-1.2-propanedione-2-oxime (in ethanol) and heating at 100 °C for 45 min. Arginase activity was expressed as ␮g urea/mg protein.

Western blotting The protein concentrations in all of the brain samples were equalized to 2 mg/ml. Tissue homogenates were then mixed with gel loading buffer (50 mM Tris–HCl, 10% sodium dodecyl sulfate (SDS), 10% glycerol, 10% 2-mercaptoethanol, 2 mg/ml bromophenol blue) in a ratio of 1:1 and then boiled for 5 min. For each brain region, the tissues from the two groups were processed at the same time. Ten microlitres of each sample was loaded in each well on a 4 –12% (for nNOS, eNOS, and arginase I) or 10% (for arginase II) Bis–Tris Criterion gel (Bio-Rad), and the proteins were transferred to polyvinylidene-difluoride (PVDF) membranes using a transblotting apparatus (Bio-Rad). The transfer was performed overnight in transfer buffer (25% methanol, 1.5% glycine, and 0.3% Tris-base). Pre-stained protein markers (41.5–203 kDa; BioRad) were always run on the same gel. Non-specific IgG binding was blocked by incubation with 5% dried milk protein and 0.1% bovine serum albumin for at least 7 h. The membranes were then incubated with an affinity-purified monoclonal mouse antibody raised against nNOS (Santa Cruz Biotechnology, sc-5302, 1:2500) or arginase I (Santa Cruz Biotechnology, sc-166920, 1:800), and polyclonal rabbit antibody raised against eNOS (Santa Cruz Biotechnology, sc-653, 1:10,000) or arginase II (1:20,000; generously provided by Dr. Tomomi Gotoh) overnight at 4 °C. The arginase II antibody was raised in a rabbit by injection of a synthetic peptide corresponding to rat arginase II (GenBank accession number: U90887) C-terminal portion (279I–298C) (Ozaki et al., 1999). The secondary antibody was an anti-mouse (Santa Cruz Biotechnology, sc-2005, 1:5000) or an anti-rabbit (Santa Cruz Biotechnology, sc-2004, 1:5000) IgG linked to horseradish peroxidase. In order to ensure that the same amount of protein was loaded in each lane, an IgG monoclonal antibody against ␤-actin (Santa Cruz Biotechnology, sc-47778, 1:20,000; secondary: goat anti-mouse IgG, Santa Cruz Biotechnology, sc-2005, 1:5000) was used as a loading control. Immunodetection was performed using the enhanced chemiluminescence (ECL) system (Amersham Biosciences, New Zealand). Hyperfilms (Amersham Biosciences, New Zealand) were analyzed by densitometry to determine the quantity of protein expressed in each group using the Bio-Rad Quantity One software (Liu et al., 2003a,b, 2004a,b,

2005). Results were expressed as volume of the band (optical density⫻area of the band) and normalized by the corresponding ␤-actin loading controls.

Amino acid and polyamine analyses Determination of arginine, citrulline, ornithine, glutamate, and GABA were carried out by the HPLC method as we have previously described (Gupta et al., 2009; Liu et al., 2009b). For each brain region, samples from the two groups were assayed at the same time and the order was counterbalanced. High-purity external and internal standards were used (Sigma, Sydney, Australia). All other chemicals were of analytical grade. After adding internal standard (trazodone) to 30 ␮l of the perchloric acid extracts, the samples were alkalized with potassium hydrogen carbonate solution (pH 9.8) and derivatized with dansyl chloride in dark at 80 °C for 20 min. The reaction was stopped by adding 10 ␮l of acetic acid followed by centrifugation at 10,000 rpm for 10 min. Forty microlitres of the supernatant was injected onto the HPLC system, which consisted of a programmed solvent delivery system at a flow rate of 1.0 ml/min, an autosampler, a reversed-phase C18 column, and a UV detector set at a wavelength of 218 nm. Identifications of arginine, citrulline, ornithine, glutamate, and GABA were accomplished by comparing the retention times of samples with the known standards. Assay validation showed that the analytical method was sensitive and reliable with acceptable accuracy (92–107% of true values) and precision (intra- and interassay CV ⬍15%). The concentrations of arginine, citrulline, ornithine, glutamate, and GABA in tissue were calculated with reference to the peak area of external standards and values were expressed as ␮g/g wet tissue. Determination of spermidine and spermine was carried out according to the HPLC method we have described previously (Gupta et al., 2009; Liu et al., 2008c). High-purity spermidine, spermine, and internal standard (1,7-diaminoheptane) were used (Sigma, Sydney, Australia). All other chemicals were of analytic grade. Briefly after adding internal standard (1, 7-diaminoheptane) to 20 ␮l of the perchloric acid extracts, the samples were alkalized with saturated sodium carbonate and derivatized with dansyl chloride. Spermidine, spermine, and internal standard were extracted with toluene. The toluene phase was evaporated to dryness, reconstituted and injected onto the HPLC system, which consisted of a programmed solvent delivery system at a flow rate of 1.5 ml/min, an autosampler, a reversed-phase C18 column, and a fluorescence detector set at the excitation wavelength of 252 nm and emission wavelength of 515 nm. Identifications of spermidine and spermine were accomplished by comparing the retention times of samples with the known standard. Assay validation showed that the analytical method was sensitive and reliable with acceptable accuracy (88 –112% of true values) and precision (intra- and inter-assay CV ⫾15%). The concentrations of spermidine and spermine in tissue were calculated with reference to the peak area of external standards and values were expressed as ␮g/g wet tissue. Agmatine and putrescine concentrations were measured by a highly sensitive LC/MS/MS method (Gupta et al., 2009; Liu et al., 2008a,b,c, 2009a). High-purity agmatine and putrescine and internal standard (1,7-diaminoheptane) were used (Sigma, Sydney, Australia). All other chemicals were of analytic grade. After adding the internal standard to 20 ␮l of the perchloric acid extracts, the samples were alkalized with saturated sodium carbonate and derivatized with dansyl chloride. Agmatine, putrescine, and internal standard were extracted with toluene. The toluene phase was evaporated to dryness, reconstituted and injected onto the LC/ MS/MS system. The samples were analyzed by a reversed-phase C18 column (150⫻2.0 mm, 5 mm, Phenomenex) with 80% acetonitrile:20% water containing 0.1% formic acid as mobile phase at a flow rate of 0.2 ml/min. The retention times of agmatine, putrescine, and the internal standard were 1.7, 4.0, and 4.8 min,

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respectively. The total run time was 15 min. Detection by MS/MS used an electrospray interface (ESI) in positive ion mode. The standard curves for agmatine and putrescine were linear up to 1000 ng/ml (R2⬎0.99). The intra- and inter-day coefficients of variation were ⬍15%. The concentrations of agmatine and putrescine in tissue were calculated with reference to the peak area of the external standards and values were expressed as ␮g/g wet tissue.

Statistical analysis Behavioral and neurochemical data were analyzed using either Student t-test or two-way repeated measures analysis of variance (ANOVA) followed by Bonferroni post hoc tests (Zolman, 1993). The significance level was set at 0.05 for all comparisons. All calculations were performed with the GraphPad Prism program. As we measured the levels of nine inter-related neurochemical variables (arginine, citrulline, ornithine, agmatine, putrescine, spermidine, spemine, glutamate, and GABA) in the sub-regions of the hippocampus and prefrontal cortex, cluster analyses were performed for each group and each brain region to determine which neurochemical variables varied closely with one another using Minitab 15. Agglomerative cluster analyses were used on the correlation coefficient distance, and comparisons of the different kinds of cluster analyses suggested that Complete linkage, McQuitty linkage, Average linkage, and Ward linkage all produced similar results (data not shown). Therefore, Ward linkage was used for all of the analyses, and dendrograms were generated using Minitab 15 (Liu et al., 2010).

RESULTS Histological results The cannula tracks were easily identified. For all of the animals, a cannula had been correctly implanted into each lateral ventricle. Behavioral results Fig. 2 presents animals’ performance in the open field. There were significant differences between the A␤35–25 and A␤25–35 groups in terms of the duration of wall-supported rearings (t(14)⫽2.68, P⬍0.05; Fig. 2A) and groomings (t(14)⫽2.34, P⬍0.05; Fig. 2B), with the latter reared less and groomed more. There were no significant differences between the two groups in terms of the path length traveled (t(14)⫽0.31, P⫽0.77; Fig. 2C), the percentage of maze used (A␤35–25: 83.31⫾4.32%, A␤25–35: 82.64⫾ 4.94%; t(14)⫽0.10, P⫽0.92), the percentage of time moving in the apparatus (A␤35–25: 69.37⫾2.78%, A␤25–35: 70.08⫾2.32%; t(14)⫽0.20, P⫽0.85), and the percentage of time spent in the outer zone (A␤35–25: 91.41⫾1.69%, A␤25–35: 93.75⫾2.67%; t(14)⫽0.74, P⫽0.47), middle zone (A␤35–25: 8.22⫾1.63%, A␤25–35: 5.63⫾2.32%; t(14)⫽0.92, P⫽0.37), and center (A␤35–25: 0.37⫾0.15%, A␤25–35: 0.57⫾0.32%; t(14)⫽0.56, P⫽0.58). Neurochemical results NOS and arginase activities and protein expression. Fig. 3A illustrates total NOS activities in the sub-regions of the hippocampus and the prefrontal cortex in the A␤35–25 and A␤25–35 groups. A significantly decreased total NOS activity was found in the CA2/3 (t(14)⫽3.14, P⬍0.01), but

Fig. 2. Animals’ performance in the open field. Mean (⫾SEM) duration (in seconds) of wall-supported rearings (A) and groomings (B), and path length (in meter) traveled in the apparatus (C), made by the A␤35–25 and A␤25–35 groups (n⫽8 in each group). The A␤25–35 group reared significantly shorter and groomed longer as compared to the A␤35–25 one. There was no significant difference between groups in the path length measurement. * Significant difference between groups at P⬍0.05.

not CA1 (t(14)⫽0.29, P⫽0.78), DG (t(14)⫽0.24, P⫽0.82), or PFC (t(14)⫽0.61, P⫽0.55), in the A␤25–35 group relative to the A␤35–25 one. For both the A␤35–25 and A␤25–35 groups, iNOS activity was not detected in any region examined. When the total arginase activity was analyzed, a significantly increased activity was found in the CA2/3 (t(14)⫽2.15, P⬍0.05), but not CA1 (t(14)⫽1.16, P⫽0.26), DG (t(14)⫽0.57, P⫽0.58), or PFC (t(14)⫽0.52, P⫽0.61), in the A␤25–35 group relative to the A␤35–25 one (Fig. 3B). Because there were differences in total NOS and arginase activities between the A␤35–25 and A␤25–35 groups in the CA2/3 sub-region of the hippocampus, we further investigated the protein levels of nNOS, eNOS (but not iNOS, as its activity was not detectable), arginase I, and

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Fig. 3. Mean (⫾SEM) NOS activity (A), arginase activity (B), and protein levels of nNOS and eNOS (C), and arginase I and arginase II (D) in the CA1, CA2/3, and DG sub-regions of the hippocampus and the prefrontal cortex (PFC) in the A␤35–25 and A␤25–35 groups (n⫽8 in each group). (E) An example of Western blot showing nNOS, eNOS, arginase I, or arginase II protein expression in CA2/3 in both groups. There were significantly decreased NOS activity and eNOS protein expression, but increased arginase activity and arginase II protein expression, in hippocampal CA2/3 in the A␤35–25 group as compared to the A␤35–25 group. * Indicates a significant difference between groups at * P⬍0.05, and ** P⬍0.01.

arginase II in this brain region, as well as hippocampal DG as a comparison. Western blotting revealed significantly decreased eNOS protein level (t(14)⫽2.71, P⬍0.05, Fig. 3C, E) and increased arginase II protein level (t(14)⫽3.57, P⬍0.01, Fig. 3D, E) in the CA2/3 in the A␤25–35 group relative to the A␤35–25 one, with no changes in terms of nNOS (t(14)⫽0.95, P⫽0.36; Fig. 3C, E) and arginase I (t(14)⫽0.55, P⫽0.59; Fig. 3D, E). However, there were no significant differences between groups in nNOS (t(14)⫽0.23, P⫽0.82; Fig. 3C), eNOS (t(14)⫽0.34, P⫽0.97; Fig. 3C), arginase I (t(14)⫽0.19, P⫽0.85; Fig. 3D), and arginase II (t(14)⫽1.02, P⫽0.33; Fig. 3D). Amino acids. Fig. 4 illustrates the tissue concentrations of arginine, citruline, ornithine, glutamate and GABA, as well as the glutamate/GABA molar ratio, in the subregions of the hippocampus and the prefrontal cortex in the A␤35–25 and A␤25–35 groups. Arginine level was similar between the two groups for each brain region (CA1: t(14)⫽0.03, P⫽0.98; CA2/3: t(14)⫽0.28, P⫽0.78; DG:

t(14)⫽0.34, P⫽0.74; PFC: t(14)⫽0.17, P⫽0.87; Fig. 4A). There was also no significant difference between the two groups in citrulline level in each brain region examined (CA1: t(14)⫽0.09, P⫽0.93; CA2/3: t(14)⫽0.10, P⫽0.92; DG: t(14)⫽1.99, P⫽0.07; PFC: t(14)⫽1.14, P⫽0.27; Fig. 4B). For ornithine, a significantly increased level was found in the CA2/3 (t(14)⫽3.14, P⬍0.01), but not CA1 (t(14)⫽0.26, P⫽0.80), DG (t(14)⫽0.29, P⫽0.78), or PFC (t(14)⫽1.50, P⫽0.16), in the A␤25–35 group relative to the A␤35–25 one (Fig. 4C). There was a significantly increased level of glutamate in the DG (t(14)⫽2.65, P⬍0.05), but not CA1 (t(14)⫽0.19, P⫽0.85), CA2/3 (t(14)⫽0.72, P⫽0.48), or PFC (t(14)⫽0.66, P⫽0.52), in the A␤25–35 group as compared to the A␤35–25 one (Fig. 4D). GABA level was similar between the two groups for each brain region (CA1: t(14)⫽0.27, P⫽0.79; CA2/3: t(14)⫽0.69, P⫽0.50; DG: t(14)⫽1.37, P⫽0.19; PFC: t(14)⫽0.13, P⫽0.90; Fig. 4E). When the glutamate/GABA molar ratio was analyzed, a significantly increased level was found in the DG

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Fig. 4. Mean (⫾SEM) arginine (A), citrulline (B), ornithine (C), glutamate (D), and GABA (E) levels and glutamate/GABA molar ratios (F) in the CA1, CA2/3, and DG sub-regions of the hippocampus and the PFC in the A␤35–25 and A␤25–35 groups (n⫽8 in each group). There were significantly increased ornithine level in the CA2/3, and increased glutamate level and glutamate/GABA molar ratio in the DG, in the A␤35–25 group as compared to the A␤35–25 group. * Indicates a significant difference between groups at * P⬍0.05, and ** P⬍0.01.

(t(14)⫽2.99, P⬍0.01), but not CA1 (t(14)⫽0.88, P⫽0.39), CA2/3 (t(14)⫽0.06, P⫽0.96), or PFC (t(14)⫽1.10, P⫽ 0.29), in the A␤25–35 group relative to the A␤35–25 one (Fig. 4F).

t(14)⫽1.54, P⫽0.15; DG: t(14)⫽1.15, P⫽0.27; PFC: t(14)⫽1.17, P⫽0.26; Fig. 5D).

Polyamines. Fig. 5 presents the tissue concentrations of agmatine, putrescine, spermidine, and spermine in the sub-regions of the hippocampus and the prefrontal cortex in the A␤35–25 and A␤25–35 groups. Agmatine levels were significantly decreased in the DG (t(14)⫽2.29, P⬍0.05) and PFC (t(14)⫽2.34, P⬍0.05), but not CA1 (t(14)⫽0.65, P⫽0.53) and CA2/3 (t(14)⫽0.76, P⫽0.46), in the A␤25–35 group relative to the A␤35–25 one (Fig. 5A). For putrescine, significantly increased levels were found in the CA2/3 (t(14)⫽2.65, P⬍0.05) and DG (t(14)⫽2.45, P⬍0.05), but not CA1 (t(14)⫽1.64, P⫽0.12) and PFC (t(14)⫽0.37, P⫽0.72), in the A␤25–35 group relative to the A␤35–25 one (Fig. 5B). There were no significant differences between groups in the levels of spermidine (CA1: t(14)⫽0.14, P⫽0.89; CA2/3: t(14)⫽0.60, P⫽0.56; DG: t(14)⫽1.10, P⫽0.29; PFC: t(14)⫽0.42, P⫽0.68; Fig. 5C) and spermine (CA1: t(14)⫽0.71, P⫽0.49; CA2/3:

Cluster analyses of the A␤35–25 rats’ data suggested that agmatine and putrescine tended to vary together, whereas arginine, citrulline, spermine, glutamate, GABA, ornithine, and spermidine tended to co-vary, in the CA1 (Fig. 6A), CA2/3 (Fig. 6C), and DG (Fig. 6E) sub-regions of the hippocampus. The most discrete clusters consisted of (1) agmatine and putrescine (for CA1, CA2/3, and DG); (2) spermidine and spermine (for DG); (3) ornithine and spermidine (for CA1 and CA2/3); (4) glutamate and GABA (for CA1 and DG); (5) citrulline and glutamate (for CA2/3), or spermine (for CA1). In the PFC, cluster analysis suggested that agmatine, ornithine, spermidine, and spermine tended to vary together, whereas arginine, citrulline, glutamate, GABA, and putrescine tended to co-vary (Fig. 6G). The most discrete clusters consisted of (1) agmatine and spemine; (2) ornithine and spermidine; (3) citrulline and GABA.

Cluster analyses

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Fig. 5. Mean (⫾SEM) agmatine (A), putrescine (B), spermidine (C), and spermine (D) levels in the CA1, CA2/3, and DG sub-regions of the hippocampus and the PFC in the A␤35–25 and A␤25–35 groups (n⫽8 in each group). There were significantly decreased agmatine levels in the DG and PFC, and increased putrescine levels in the CA2/3 and DG, in the A␤35–25 group as compared to the A␤35–25 group. * Significant difference between groups at P⬍0.05.

Cluster analyses of the A␤25–35 rats’ data revealed that the pattern of the clusters was different in each brain region examined when compared to the A␤25–35 group. In CA1, for example, cluster 1 was putrescine, whereas cluster 2 consisted of the remaining eight neurochemical variables, with the most discrete clusters being (1) arginine and ornithine; (2) citrulline and glutamate; and (3) spermidine and spermine (Fig. 6B). In CA2/3, arginine, spermidine, agmatine, and putrescine tended to vary together, whereas citrulline, ornithine, GABA, glutamate, and spermine tended to co-vary, with the most discrete clusters consisting of (1) arginine and spermidine; (2) agmatine and putrescine; (3) ornithine and GABA; and (4) glutamate and spermine (Fig. 6D). In DG, citrulline, putrescine, and agmatine tended to vary together, whereas agmatine, spermidine, spermine, ornithine, glutamate, and GABA tended to co-vary, with the most discrete clusters being (1) citrulline and putrescine; (2) spermidine and spermine; and (3) glutamate and GABA (Fig. 6F). In the PFC, ornithine and putrescine tended to vary together, whereas arginine, GABA, spermidine, citrulline, glutamate, agmatine, and spermine tended to co-vary (Fig. 6H). The most discrete clusters consisted of (1) arginine and GABA; (2) citrulline and glutamate; (3) agmatine and spemine; and (4) ornithine and putrescine. These results suggest that A␤25–35 has a major effect on the way that these variables co-vary.

DISCUSSION Previous research has shown that a single i.c.v. infusion of pre-aggregated A␤25–35 produces behavioral deficits (for a review see Gulyaeva and Stepanichev, 2010). In the present study, animals that received the i.c.v. infusion of

A␤25–35 (30 nmol/rat) displayed less wall-supported rearings, more groomings, and no change in terms of the path length traveled, the percentage of maze used, the percentage of time moving, and the percentage of time spent in the outer zone of the open-field apparatus relative to those that received the same dose of A␤35–25 at the time point of 6 days after A␤ infusion. These findings suggest that A␤25–35 results in reduced exploratory activity with no effect on locomotion. Grooming is a multifunctional behavior that can be elicited by novelty and other stressors (for a review see Spruijt et al., 1992). Either a positive or negative relationship between grooming behavior and anxiety has been documented previously (Mondragón et al., 1987; van Erp et al., 1994; D’Aquila et al., 2000; Homberg et al., 2002). Previous studies reported that A␤25–35 treatment increases, reduces, or does not affect animals’ anxiety levels (Olariu et al., 2001; Stepanichev et al., 2006; Yenkoyan et al., 2009). Bergin and Liu (2010) found no difference in animals’ performance on the open field when tested at 4 weeks after i.c.v. infusion of A␤25–35 or A␤35–25 (30 nmol/rat). The present study, for the first time, investigated how A␤25–35 affected arginine metabolism in the sub-regions of the hippocampus and the prefrontal cortex. At the time point of 8 days after A␤ infusion, a single bilateral i.c.v. infusion of pre-aggregated A␤25–35 resulted in decreased total NOS activity and increased total arginase activity in the CA2/3, but not other regions examined. As NOS and arginase share the substrate arginine, these changes reflect the competitive relationship between the two enzymes. It should be pointed out that iNOS activity was not detectable in the present study. However, Alkam et al.

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Fig. 6. Dendrograms showing the similarities in the degree of expression of the nine neurochemical variables (arginine, citrulline, ornithine, agmatine, putrescine, spermidine, spermine, glutamate, and GABA) in the CA1 (A, B), CA2/3 (C, D), DG (E, F) sub-regions of the hippocampus and the PFC (G, H) in the A␤35–25 (A, C, E, G) and A␤25–35 (B, D, F, H) groups (n⫽8 in each group). For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

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(2008) reported that A␤25–35 (3 ␮g/3 ␮l, i.c.v.) resulted in increased iNOS mRNA levels in the mouse hippocampus at 2 h to 5 days after i.c.v. injection. Because the longest time point tested was 5 days post-injection in the study of Alkam et al. (2008), it is unclear whether iNOS mRNA level returns to the basal level on day 8 (the time point of the present study). In order to understand which isoforms of NOS and arginase contribute to the enzyme activity changes described above, Western blot was conducted to measure the protein levels of nNOS and eNOS (but not iNOS, as its activity was not detectable), as well as arginase I and II, in hippocampal CA2/3 and DG (as a comparison). Significantly decreased eNOS (but not nNOS) and increased arginase II (but not arginase I) protein levels were found in the CA2/3, but not DG, in the A␤25–35 group relative to the A␤35–25 one, suggesting that eNOS and arginase II are responsible for A␤25–35-induced changes in total NOS and arginase activities, respectively. It has been documented that NO derived from eNOS is a key factor for the stabilization and regulation of the vascular microenvironment (de la Torre, 2009). In AD brains, NFTs and SPs are associated with reduced capillary expression of eNOS (Provias and Jeynes, 2008; Jeynes and Provias, 2009). We have recently observed significantly reduced total NOS activity and eNOS protein expression in the superior frontal gyrus and hippocampus in the AD brains (Liu et al., unpublished observation). A number of studies have demonstrated that A␤ induces endothelial apoptosis and dysfunction (Yin et al., 2005; Chisari et al., 2010), which supports the present finding. Although the effects of oligomeric A␤ on arginase have not been investigated previously, increased arginase I and arginase II mRNA levels have been found in the AD brains (Colton et al., 2006; Hansmannel et al., 2010). Furthermore, the presence of the rare arginase II allele rs742869 was associated with an increase in the risk of AD and an earlier age-at-onset (Hansmannel et al., 2010). It has been shown that arginase II is expressed in the vascular endothelial cells and competes the use of arginine with eNOS, hence regulates vascular endothelial function and vascular tone through eNOS (Lim et al., 2007; for a review see Durante et al., 2007). The present study further investigated the effects of A␤25–35 on arginine metabolism by measuring the tissue concentrations of arginine and its metabolites in the subregions of the hippocampus and the prefrontal cortex. A␤25–35 did not affect the levels of arginine and citrulline (the by-product of NO) in all four brain regions but resulted in significantly increased level of ornithine (the product of arginase) in the CA2/3 that parallels with A␤25–35-induced increase in arginase activity in this region. Although NOS activity was significantly decreased in the CA2/3 in the A␤25–35 group, there was no change in the citrulline level, indicating no parallel relationship between NOS and citrulline (see also Liu et al., 2009b). It has been shown that citrulline can be formed from ornithine by ornithine transcarbamylase in addition to arginine by NOS, but it can also be recycled to generate arginine by argininosuccinate

synthetase and argininosuccinate lyase (Wu and Morris, 1998; Wiesinger, 2001; Morris, 2006). The major route of the polyamine production in mammalian cells is de novo synthesis from ornithine by ODC. It has been documented that polyamines are essential for cell proliferation and differentiation, synthesis of DNA, RNA and proteins, protein phosphorylation, signal transduction, as well as the regulation of neurotransmitter receptors (for reviews see Williams, 1997; Wallace, 2000; Oredsson, 2003). Among the three polyamines, putrescine has a negative influence on the N-methyl-D-aspartate (NMDA) receptor function, whereas spermidine and spermine are the positive modulators of NMDA receptors (Williams et al., 1994; Rock and Macdonald, 1995; Williams, 1997). In the present study, the i.c.v. infusion of pre-aggregated A␤25–35 resulted in increased levels of putrescine in the CA2/3 (which parallels with increased ornithine level in this region) and DG, increased glutamate level and the glutamate/GABA ratio in the DG, with no effects on spermidine, spermine, and GABA. It is unclear from the present findings what kind of glutamate pools are affected by A␤25–35 in DG, as we only measured the tissue content of glutamate. Harkany et al. (2000) reported that A␤ infusion resulted in increased extracellular concentrations of glutamate in the rat magnocellular nucleus basalis. The findings of the present study suggest A␤25–35-induced imbalance in glutamate-GABA level in hippocampal DG, hence altered excitatory activity due to the over-activation of NMDA receptors (Wu and Hou, 2010; for a review see Kaminsky et al., 2010), which is consistent with the reports of pathogenic A␤ assemblies-elicited aberrant excitatory activity in the DG (Palop et al., 2007; Harris et al., 2010). Given the negative influence of putrescine on the NMDA receptor function, it is likely that increased putrescine level in DG may be a compensatory mechanism to maintain the normal excitability of granule cells. Malaterre et al. (2004) demonstrated that the reduction in putrescine levels significantly impaired adult neurogenesis in hippocampal DG in young rats, suggesting an important role of putrescine in hippocampal neurogenesis. Since the i.c.v. infusion of preaggregated A␤25–35 impairs hippocampal neurogenesis (Li and Zuo, 2005; Li et al., 2010), it is also possible that increased putrescine level may be a compensatory/protective mechanism to maintain the normal rate of new born cells in the DG. Agmatine is the product of ADC, and regulates the NO production by influencing the activity of three isoforms of NOS (for a review see Halaris and Plietz, 2007). In addition to being a precursor of putrescine (through agmatinase), agmatine regulates the intracellular content of polyamines through the induction of antizyme, a small regulatory protein that inhibits ODC and downregulates polyamine uptake (Satriano, 2003). There is evidence suggesting that endogenous agmatine may directly participate in the processes of learning and memory as a novel neurotransmitter (Liu et al., 2008a, 2009a; Leitch et al., 2011). In the present study, the i.c.v. infusion of pre-aggregated A␤25–35 resulted in decreased levels of agmatine in hippocampal DG and the prefrontal cortex at the time point of 8 days

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after A␤ infusion. It is of interest to mention that agmatine (40 mg/kg) administered intraperitoneally 30 min prior to A␤25–35 i.c.v. infusion and once daily for a further nine consecutive days significantly protects against A␤25–35induced learning and memory deficits (Bergin and Liu, 2010). Hence, reduced levels of endogenous agmatine in the hippocampus and prefrontal cortex may have significant functional significance. Future research is required to further explore the therapeutic potential of exogenous agmatine in the prevention/treatment of behavioral deficits associated with A␤ and AD. In the present study, cluster analyses were performed to determine if the nine related neurochemical variables (arginine, citrulline, ornithine, agmatine, putrescine, spermidine, spermine, glutamate, and GABA) clustered into distinct groups and whether this changed as a function of A␤25–35. Overall, in the A␤35–25 rats the clusters were similar across the CA1, CA2/3, and DG sub-regions of the hippocampus, but not the prefrontal cortex, suggesting that the interplay among the nine neurochemical variables may have a region-specific pattern. For the A␤25–35 rats, however, the clusters were quite different when compared to the A␤35–25 group across all four brain regions examined, suggesting the effects of A␤25–35 on the way that these variables co-vary. In terms of the known neurobiology, chemically related spermidine and spermine varied together, albeit in different clusters. Arginine and citrulline or ornithine also co-varied, which would be expected given that NOS and arginase produces citrulline and ornithine from L-arginine, respectively. Since GABA is derived from glutamate, it would also be expected that these two neurotransmitters would co-vary. The relationship between agmatine and putrescine, which is apparent particularly in the sub-regions of the hippocampus, is interesting. It has been well documented that agmatine can be converted to putrescine by agmatinase and regulates the intracellular content of polyamines through its influence on ODC and polyamine uptake (Wu and Morris, 1998; Satriano, 2003; Halaris and Plietz, 2007). It should be pointed out that in the present study animals had behavioral testing experience prior to the tissue harvest. Hence, one may argue that the neurochemical changes observed may not reflect the effects of A␤25–35 on basal parameters due to stress induced by behavioral testing. A number of studies have reported that stress affects NOS expression and agmatine levels in the brain. Keser et al. (2011), for example, reported that forced swimming stress significantly increased nNOS expression in the hippocampus in the female, but not male (the case for the present study), rats. Khovryakov et al. (2010) found that chronic stress induced significant increases in nNOS and iNOS expression in many parts of the brain (predominantly in the neocortex and hippocampus). Zhu et al. (2008) demonstrated that immobilization led to significantly increased endogenous agmatine levels in many brain regions (including the prefrontal cortex and hippocampus). Given the region-specific effects observed in the present study, it is most likely that the neurochemical changes are induced by pre-aggregated A␤25–35.

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CONCLUSION The present study demonstrated that a single bilateral i.c.v. infusion of pre-aggregated A␤25–35 produced behavioral deficits and altered arginine metabolism at the time point of 1 week after A␤ infusion in a region-specific manner. Within the hippocampus, the CA2/3 and DG, but not CA1, appeared to be affected greatly. It has been shown that the DG functions as a “filter/gate” to prevent excessive excitatory input entering CA3, which has an important role in integrating various types of information and is highly susceptible to epileptiform activity (Amaral and Witter, 1995; Christian and Dudek, 1988; Lisman, 1999). A number of studies have reported cell loss in the CA1 region at the time points of 4 –10 weeks following the infusion/injection of pre-aggregated A␤25–35 (Stepanichev et al., 2004, 2006; Limón et al., 2009a,b). It is, therefore, possible that the CA1 region may show neurochemical changes at these later time points. Given the increasing evidence suggesting prominent role of arginine and its metabolites in AD pathogenesis (for reviews see Law et al., 2001; Malinski, 2007; Yi et al., 2009), there is a need to better understand how arginine metabolic profile changes in the hippocampus in transgenic models of AD and AD patients. Acknowledgments—We would like to thank Dr. Tomomi Gotoh for providing arginase II antibody. This work was supported by the Department of Anatomy and Structural Biology, University of Otago. S.A.C. was a recipient of the Allan Wilkinson Summer Studentship Scholarship, Otago Medical Research Foundation.

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(Accepted 22 July 2011) (Available online 27 July 2011)