Behavioural Brain Research 235 (2012) 263– 272

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Behavioural Brain Research

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Research report

Neuroprotective effects of agmatine in mice infused with a single intranasaladministration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Filipe C. Matheusa, Aderbal S. Aguiar Jr. a,b, Adalberto A. Castrob, Jardel G. Villarinhoc, Juliano Ferreirac,Cláudia P. Figueiredod, Roger Walze, Adair R.S. Santos f, Carla I. Tascab, Rui D.S. Predigera,∗

a Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, UFSC, 88049-900 Florianópolis, SC, Brazilb Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, UFSC, 88040-900 Florianópolis, SC, Brazilc Departamento de Química, Universidade Federal de Santa Maria, UFSM, Santa Maria, RS 97105-900, Brazild Departamento de Fármacos, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, UFRJ, Rio de Janeiro, RJ, Brazile Departamento de Clínica Médica, Hospital Universitário, Universidade Federal de Santa Catarina, UFSC, Florianópolis, SC, Brazilf Laboratório de Neurobiologia da Dor e Inflamac ão, Departamento de Ciências Fisiológicas, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, UFSC,88040-900 Florianópolis, SC, Brazil

h i g h l i g h t s

! Agmatine increased the survival rate of aging mice infused intranasally with MPTP.! Agmatine improved social memory and motor impairments induced by i.n. MPTP administration.! Agmatine protected against the dopaminergic cell loss induced by i.n. MPTP administration.! Agmatine prevented MPTP-induced decrease of hippocampal glutamate uptake in aging mice.! Agmatine may represent a new therapeutic tool for the management of cognitive and motor symptoms of Parkinson’s disease.

a r t i c l e i n f o

Article history:Received 24 May 2012Received in revised form 9 August 2012Accepted 12 August 2012Available online 17 August 2012

Keywords:AgmatineParkinson’s disease1-Methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP)IntranasalAging miceNon-motor symptoms

a b s t r a c t

We have recently demonstrated that rodents treated intranasally with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) suffered impairments in olfactory, cognitive, emotional and motor functionsassociated with time-dependent disruption of dopaminergic neurotransmission in different brain struc-tures conceivably analogous to those observed during different stages of Parkinson’s disease (PD).Agmatine, an endogenous arginine metabolite, has been proposed as a novel neuromodulator that playsprotective roles in several models of neuronal cellular damage. In the present study we demonstratedthat repeated treatment with agmatine (30 mg/kg, i.p.) during 5 consecutive days increased the sur-vival rate (from 40% to 80%) of 15-month-old C57BL/6 female mice infused with a single intranasal (i.n.)administration of MPTP (1 mg/nostril), improving the general neurological status of the surviving animals.Moreover, pretreatment with agmatine was found to attenuate short-term social memory and locomo-tor activity impairments observed at different periods after i.n. MPTP administration. These behavioralbenefits of exogenous agmatine administration were accompanied by a protection against the MPTP-induced decrease of hippocampal glutamate uptake and loss of dopaminergic neurons in the substantianigra pars compacta of aging mice, without altering brain monoamine oxidase B (MAO-B) activity. Theseresults provide new insights in experimental models of PD, indicating that agmatine represents a poten-tial therapeutic tool for the management of cognitive and motor symptoms of PD, together with itsneuroprotective effects.

© 2012 Elsevier B.V. All rights reserved.

∗ Corresponding author at: Departamento de Farmacologia, Universidade Federalde Santa Catarina, Campus Trindade, 88049-900 Florianópolis, SC, Brazil.Tel.: +55 48 3721 9491; fax: +55 48 3337 5479.

E-mail address: ruidsp@hotmail.com (R.D.S. Prediger).

1. Introduction

Parkinson’s disease (PD) is a debilitating disease primarilycharacterized by the progressive loss of neuromelanin-containingdopaminergic neurons in the substantia nigra pars compacta (SNpc)with presence of eosinophillic, intracytoplasmic, proteinaceousinclusions termed as Lewy bodies and dystrophic Lewy neuritesin surviving neurons [37]. At the time of diagnosis, patients typi-cally display an array of motor impairments including bradykinesia,

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264 F.C. Matheus et al. / Behavioural Brain Research 235 (2012) 263– 272

resting tremor, rigidity, and postural instability. Although most ofthe typical motor impairments are due to the loss of nigrostriataldopaminergic neurons, PD affects multiple neuronal systems bothcentrally and peripherally, leading to a constellation of non-motorsymptoms including olfactory deficits, affective disorders, memoryimpairments, as well as autonomic and digestive dysfunction [13].These non-motor features of PD do not meaningfully respond todopaminergic medication and are a challenge to the clinical man-agement of PD [13].

Numerous epidemiological and experimental studies suggestthat exposure to agricultural chemicals, viruses, metals, and othertoxins contribute to its pathogenesis (for review see [19,56]).In some cases such agents conceivably enter the brain via theolfactory neuroepithelium, a concept termed the olfactory vectorhypothesis [22,56]. In this context, we have recently proposed anew experimental model of PD consisting of a single intranasal(i.n.) administration of the proneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in rodents [12,50,57–59]. Youngadult Wistar rats and C57BL/6 mice (3–6-months-old) treatedintranasally with MPTP suffer impairments in olfactory, cognitive,emotional and motor functions conceivably analogous to thoseobserved during different stages of PD. Such infusion causes time-dependent loss of tyrosine hydroxylase (TH) in the olfactory bulband SNpc, resulting in significant dopamine depletion in differ-ent brain areas [57–59]. We have also identified some pathogenicmechanisms possibly involved in the neurodegeneration inducedby i.n. administration of MPTP including mitochondrial dysfunc-tion, oxidative stress, activation of apoptotic cell death mechanismsand glutamatergic excitotoxicity (for review see [57]). Therefore,the i.n. MPTP administration seems to represent a valuable rodentmodel for testing novel drugs for both motor and non-motor symp-toms relief as well as the discovery of compounds to modify thecourse of PD.

On the other hand, there is increasing evidence that alter-ations in glutamatergic neurotransmission have a pivotal role inthe pathophysiology of PD [10,63]. For instance, dopaminergicneurons in the SNpc receive moderate excitatory glutamater-gic neurons input from the subthalamic nucleus (STN) [33]. It ispostulated that overstimulation of glutamate receptors on nigraldopaminergic neurons may be involved in the neuronal degenera-tion and progression of PD [10,63]. Moreover, it was demonstrateda pronounced increase in the extracellular levels of glutamatein the substantia nigra of mice treated chronically with MPTP[48]. Indeed, recent studies have demonstrated that d-cycloserine,a partial agonist of the glycine binding site of the N-methyl-d-aspartate (NMDA) receptor, improves mnemonic impairments andanxiety-like behaviors observed in MPTP-lesioned monkeys [65]and rats [38,69]. Finally, there are promising findings from clinical[1,23] and preclinical studies of the efficacy of the NMDA recep-tor antagonist memantine for the treatment of PD [43]. Thus, drugsmodulating the function of glutamate NMDA receptors may havebeneficial effects in PD therapy.

In this context emerges agmatine, a polyamine that is synthe-sized after decarboxylation of l-arginine by arginine decarboxylaseand it is hydrolyzed to putrescine by enzyme agmatinase and thathas been recently proposed as neuromodulator [34,61]. In the brain,agmatine is stored in synaptic vesicles [34] and is released byCa2+-dependent depolarization [61] with multiple molecular tar-gets proposed, including the binding and blockage of glutamateNMDA receptors [70]. Age-related changes in agmatine levels invarious brain structures have been described indicating potentialinvolvement of agmatine in aging process [45]. Of high impor-tance, recent studies have demonstrated the protective effectsof exogenous agmatine administration in animal models of neu-rodegenerative disorders such as PD [15,16,30] and Alzheimer’sdisease [7].

It has been well established that aging is the most prominent riskfactor for PD. However, preclinical studies addressing the behav-ioral and neurochemical effects of dopaminergic neurotoxins suchas MPTP have been widely performed in young adult animals whichmay represent one possible explanation for the limited successon translational research in PD. Therefore, the aim of the presentstudy was to evaluate the potential of the repeated administrationof exogenous agmatine to prevent behavioral and neurochemicalchanges induced by a single i.n. MPTP administration in aging mice.

2. Materials and methods

2.1. Animals

Experiments were conducted using 15-month-old female C57BL/6 mice weigh-ing 25–35 g purchased from the Multidisciplinary Center for Biological Research(CEMIB, São Paulo, Brazil). The animals were kept in collective cages (5 animals percage) and maintained in a room under controlled temperature (23 ± 1 ◦C) and 12 hlight cycle (lights on 7:00 AM), with free access to food and water. The animals weretreated, manipulated and euthanized according to the “Principles of Laboratory Ani-mal Care” (NIH publication no. 80-23, revised 1996) and approved by the Committeeon the Ethics of Animal Experiments of the Federal University of Santa Catarina(CEUA/UFSC; www.ceua.ufsc.br; protocol 23080.019002/2009-71). All efforts weremade to minimize the number of animals used and their suffering.

2.2. Drugs and treatment

The animals were allocated to the following groups: (i) control + control (n = 13),(ii) control + MPTP (n = 20), (iii) agmatine + control (n = 14) or (iv) agmatine + MPTP(n = 17). The variation in animal’s body weights was considered and counterbal-anced across the four groups. Agmatine sulfate (Sigma Chemicals Co., USA) wasfreshly dissolved in 0.9% NaCl (saline) to a final concentration of 3 mg/ml beforeeach daily treatment. Animals were administered by i.p. route once daily for 5 con-secutive days with control solution (saline) or agmatine (30 mg/kg) in a volume of0.1 ml/10 g of body weight. The dose of agmatine utilized was chosen based on previ-ous studies conducted in our laboratory [28,54,64]. One hour after the last injectionof agmatine, the animals were infused intranasally with a single bilateral dose ofMPTP (1 mg/nostril) or control solution (saline) (Fig. 1).

MPTP HCl (Sigma Chemicals Co., USA) was administered by i.n. route accordingto the procedure previously described [21] and modified in our laboratory [58].Briefly, mice were lightly anaesthetized with isoflurane 0.96% (0.75 CAM; AbbotLaboratórios do Brasil Ltda., RJ, Brazil) using a vaporizer system (SurgiVet Inc., WI,USA) and a 7 mm piece of PE-10 tubing was inserted through the nostrils. The tubingwas connected to a peristaltic pump set at a flow rate of 12.5 !l/min. The MPTP HClwas dissolved in saline at a concentration of 20 mg/ml, after which it was infused for4 min (1 mg/nostril). The control solution consisted of saline. Animals were given a1 min interval to regain normal respiratory function and then this procedure wasrepeated with infusions administered through the contralateral nostrils.

2.3. Survival analysis

The animals’ survival rate in each group was assessed throughout the experi-mental protocol period. No death was observed within the 5 days of repeated i.p.treatment with agmatine or control (data not shown). The number of deaths wasmonitoring until the 21st day after the i.n. MPTP administration for later assemblyof the survival curve.

2.4. Behavioral tests

During a period of 3–19 days after the i.n. administration of MPTP, the animalswere submitted to a battery of behavioral paradigms that included the activity cham-ber, social recognition, neurological severity score and open-field tasks (Fig. 1). Thetime point for the performance of each behavioral task was chosen based on previ-ous studies using the i.n. MPTP model [12,50,57]. All tests were carried out between9:00 and 14:00 h and they were scored by the same rater in an observation roomwhere the mice had been habituated for at least 1 h before the beginning of the tests.Behavior was monitored through a video camera positioned above the apparatusesand the images were later analyzed with the ANY Maze video tracking (StoeltingCo., Wood Dale, IL, USA) by an experienced experimenter who was unaware of theexperimental group of the animals tested.

2.4.1. Activity chambersIn order to assess early effects of MPTP on locomotor activity, the animals were

tested 3 days after i.n. MPTP administration in activity chambers. The chambers weremade of black fiberglass (50 cm × 25 cm × 15 cm) and the experiments were per-formed in a sound-attenuated room under low-intensity light (12 lux). Each mousewas placed in the center of the apparatus and the total distance traveled (m) and

F.C. Matheus et al. / Behavioural Brain Research 235 (2012) 263– 272 265

Fig. 1. Time course of behavioral and neurochemical tests following the pretreatment (during 5 consecutive days) with control (saline) or agmatine (30 mg/kg, i.p.) and asingle intranasal (i.n.) administration of control (saline) or MPTP (1 mg/nostril) in 15-month-old female C57BL/6 mice.

the average speed (m/s) were registered during 5 min. Chambers were cleaned with10% ethanol between animals.

2.4.2. Social recognitionShort-term social memory was assessed 7 days after i.n. MPTP administration

with the social recognition task previously evaluated in our laboratory [58]. Juve-nile female C57BL/6 mice (25–30 days old) served as social stimuli for the adultmice in the social recognition task. The adult animals were isolated in individualcages during seven days before the task. All juveniles were isolated in individualcages for 20 min prior to the beginning of the experiment. The social recognitiontask consisted of two successive presentations (5 min each), separated by an inter-trial interval of 30 min, where the juvenile was placed in the home cage of the adultmouse and the time spent by the adult in investigating the juvenile (nosing, sniffing,grooming, or pawing) was recorded in the two presentations. Time spent in socialinvestigation by the adult mouse was measured and then expressed for each ani-mal as the ratio of the second exposure to the first exposure (Ratio of InvestigationDuration (RID)). A reduction in RID reflects a decrease in investigation behavior dur-ing the second encounter, demonstrating the recognition ability of the adult mouse.This transformation was chosen in order to minimize day-to-day variations on thebaseline of performance and to equalize variances among different groups [12].

2.4.3. Neurological severity scoreThirteen days after i.n. administration of MPTP, mice performed the 10-point

neurological severity score (NSS), a composite behavioral scale designed to measurethe general neurological state previously described [25] and recently evaluated inour laboratory [66]. Mice were assessed for the following items: presence of paresis,inability to walk straight, impairment of seeking behavior, absence of perceptiblestartle reflex, inability to exit a 30 cm diameter circle, inability to walk on 3, 2, and1 cm wide beams, and inability to balance on a 0.7 cm-wide beam and a 0.5 cm-diameter round beam for at least 10 s. If mice showed the impairment described byan item, a value of 1 was added to total NSS score. Higher scores in the NSS indicategreater neurological impairment [66].

2.4.4. Open fieldThe spontaneous locomotor activity of the animals was evaluated in an open-

field arena at 19 days after i.n. MPTP administration. The apparatus, made of woodcovered with impermeable Formica, had a black floor of 50 cm × 50 cm (dividedby white lines into 9 squares of equal size) and transparent walls, 40 cm high. Eachmouse was placed in the center of the open field and the numbers of squares crossedand rearing were registered during 5 min. The apparatus was cleaned with ethanolsolution (10% v/v) and dried with paper towels after each trial in order to avoid odorimpregnation.

2.5. Immunohistochemistry for tyrosine hydroxylase (TH)

For the investigation of possible neuroprotective effects of agmatine against theloss of dopaminergic neurons induced by i.n. MPTP administration, five animals ofeach group were intracardially perfused with 4% paraformaldehyde in physiologi-cal saline (NaCl 0.9%) at 21 days after MPTP treatment. Brains were collected andfixed in a phosphate buffered saline (PBS) solution containing 4% paraformalde-hyde for 24 h at room temperature, dehydrated by graded ethanol, and embeddedin paraffin. Immunoreactivity of TH-positive neurons in the substantia nigra parscompacta (SNpc) was assessed on paraffin tissue sections (4 !m), using the anti-THmonoclonal antibody (1:300, catalog MAB318, Millipore/Chemicon International,

Technology, USA) as described previously [58]. Following quenching of endogenousperoxidase with 3% hydrogen peroxide in methanol for 20 min, high tempera-ture antigen retrieval was performed by immersion of the slides in a water bathat 95–98 ◦C in 10 mM trisodium citrate buffer pH 6.0, for 45 min. After overnightincubation at 4 ◦C with primary antibodies, the slides were washed with PBS andincubated with the appropriate biotinylated secondary antibody, and then pro-cessed using the Vectastain Elite ABC reagent (Vector Laboratories, Burlingame, CA,USA) according to the manufacturer’s instructions. The sections were washed in PBS,and the visualization was completed by using 3,3-diaminobenzidine (DAB) (DakoCytomation) in chromogen solution and counterstained with Harris’s hematoxylin.Tissues from the four groups were placed on the same slide and processed under thesame conditions. We included negative control that consisted in replace the primaryantibody by nonimmune serum in equivalent concentration.

The number of TH-stained positive cells in the SNpc was assessed at three levelsbetween coordinates −2.75 mm and −2.92 mm with respect to the bregma. Threealternate 4 !m paraffin sections with an individual distance of ≈60 !m of each sec-tion were obtained, and the number of TH-stained positive cells was determinedupon visual inspection at the SNpc with optical microscope (Eclipse 50I; Nikon,Melville, NY) by using a counting grid at ×400 magnification. Results were expressedas mean number of TH-stained positive cells per mm2 from three tissue sections.

2.6. l-[3H]glutamate uptake

The glutamate uptake assay was evaluated as previously described [55]. Themice were decapitated 21 days after i.n. MPTP administration and the hippocampiwere quickly removed and stored in KRB (in mM = 122 NaCl, 3 KCl, 1.3 CaCl2, 1.2MgSO4, 0.4 KH2PO4, 25 NaHCO3 and 10 d-glucose) previously aerated with carbogen(95% O2–5% CO2) to reach pH 7.4. The tissue sections (400 !m thick) were obtainedusing a tissue slicer (Mcilwain Tissue Chopper, Australia). On average, five slices fromthe middle of the hippocampus were obtained and placed in a 96-well multiwellplate containing KRB, pH 7.4. The slices were incubated for 1 h before the gluta-mate uptake assays were performed. After incubation, the hippocampal slices werewashed for 15 min at 37 ◦C in a Hank’s balanced salt solution (HBSS), composition inmM: 1.29 CaCl2, 136.9 NaCl, 5.36 KCl, 0.65 MgSO4, 0.27 Na2HPO4, 1.1 KH2PO4, and 5HEPES. Uptake was assessed using 0.33 !Ci/ml l-[3H]glutamate with 100 !m unla-beled glutamate in a final volume of 300 !l. Incubation was immediately stoppedafter 7 min by discarding the incubation medium and the slices were submitted totwo ice-cold washes with 1 ml of HBSS. The slices were solubilized by adding a solu-tion with 0.1% NaOH/0.01% SDS and incubated overnight. Aliquots of slice lysateswere taken for determination of the intracellular content of l-[3H]glutamate by scin-tillation counting. Sodium-independent uptake was determined by using cholinechloride instead of sodium chloride in the HBSS. Unspecific sodium-independentuptake was subtracted from total uptake to obtain the specific sodium-dependentglutamate uptake. Results were expressed as nmol of l-[3H]glutamate taken up permilligram of protein per minute.

2.7. Monoamine oxidase (MAO) assay

To investigate whether the treatment with agmatine may interfere with the gen-eration of the toxic metabolite 1-methyl-4-phenylpyridinium (MPP+) from MPTP,agmatine was tested for its in vitro inhibitory potential on mouse MAO-A and MAO-B activities in brain mitochondrial homogenates by a fluorometric method usingkynuramine as substrate, as previously described [46]. Brain (all regions withoutcerebellum) mitochondria from 15-month-old female C57BL/6 mice were isolated

266 F.C. Matheus et al. / Behavioural Brain Research 235 (2012) 263– 272

Fig. 2. Effects of the pretreatment (during 5 consecutive days) with control (i.p.)or agmatine (30 mg/kg, i.p.) on survival rate of 15-month-old female C57BL/6 miceinfused intranasally with control or MPTP (1 mg/nostril). The lines represent thepercentage survival animals of each group over the course of 21 days after thei.n. MPTP administration. * P < 0.05 compared to the percentage of survival of thecontrol/control group. # P < 0.05 compared to the percentage of survival of theagmatine/MPTP group (Breslow–Gehan–Wilcoxon test).

according to the method of Naoi et al. [51]. The obtained mitochondrial pellet wassuspended in 10 mM sodium phosphate buffer (pH 7.4) to 100–300 mg/mL andthen was used for assay. Briefly, assays were performed in duplicate in a finalvolume of 500 !l containing 0.5 mg of protein and incubated at 37 ◦C for 30 min.Activities of the MAO-A and MAO-B isoforms were isolated pharmacologically byincorporating 250 nM selegiline (selective MAO-B inhibitor) or 250 nM clorgyline(selective MAO-A inhibitor) into the reaction mixture. The reaction mixture (con-taining mitochondrial fractions, agmatine and inhibitors) was pre-incubated at 37 ◦Cfor 5 min and the reaction was started by addition of 50 !l of kynuramine (90 !mfor MAO-A and 60 !m for MAO-B). Agmatine was tested in a concentration rangeof 0.01–1000 !m and the results were expressed as percentage of control (tubewithout agmatine).

2.8. Statistical analysis

Data for Neurological Severity Score are shown as median (interquartilerange) and comparisons between groups were performed by Kruskal–Wallis non-parametric test followed by Dunn’s multiple comparison tests. Statistical analysisof survival curves were performed with the Cox–Mantel test (log-rank) followedby Gehan–Breslow–Wilcoxon test. The rest of data was checked for normalityof frequency distribution with the Kolmogorov–Smirnov test and expressed asmean ± standard error of mean (S.E.M.). In this case, Student’s t-test and analysis ofvariance (ANOVA) were applied when appropriate, as informed in the results sectionand figure legends. Following significant ANOVAs, multiple post hoc comparisonswere performed using the Newman–Keuls test. The accepted level of significancefor all tests was P ≤ 0.05. All tests were performed using the Statistica® softwarepackage (Stat Soft Inc., USA).

3. Results

3.1. Effects of agmatine on the survival rate of aging mice infusedintranasally with MPTP

As can be seen in Fig. 2, log-rank Mantel–Cox test followedby Gehan–Breslow–Wilcoxon test indicated a lower percentage ofsurviving animals in the control/MPTP group when compared tothe control/MPTP group (treatment factor: P ≤ 0.05). The statisticalanalysis performed point-by-point indicated an increased mortal-ity (about 50%) of MPTP-infused mice from the 1st to the 13th dayafter treatment. Remarkably, repeated treatment with agmatine(30 mg/kg, i.p.) during 5 consecutive days increased significantlythe survival rate of MPTP-treated aging mice to about 75% (Fig. 2)(interaction factor: P ≤ 0.05).

3.2. Effects of agmatine on the social recognition memory ofaging mice infused intranasally with MPTP

The results for the effects of i.n. administration of MPTP(1 mg/nostril) on the short-term social recognition memory ofaging mice pretreated with control or agmatine (30 mg/kg) areillustrated in Fig. 3. Two-way ANOVA revealed significant effects forthe pretreatment [F1,33 = 10.32, P ≤ 0.01], treatment [F1,33 = 20.72;

Fig. 3. Effects of the pretreatment (during 5 consecutive days) with control (i.p.) oragmatine (30 mg/kg, i.p.) on the social recognition memory of 15-month-old femaleC57BL/6 mice evaluated in the social recognition task 7 days after i.n. infusion ofMPTP (1 mg/nostril). Data are expressed as the mean ± S.E.M. of RIDs (i.e. the ratioof the second exposure to the first exposure) when the same juvenile was exposedfor 5 min with an interval of 30 min [control/control (n = 10); control/MPTP (n = 9);agmatine/control (n = 10); and agmatine/MPTP (n = 9)]. *P ≤ 0.05 compared to thecontrol/control group. #P ≤ 0.05 compared to the control/MPTP group (two-wayANOVA followed by Newman–Keuls test).

P ≤ 0.001] and their interaction [F1,33 = 8,44, P ≤ 0.01] in the ratio ofinvestigation duration (RID).

Post hoc comparisons indicated that the i.n. MPTP treatmentpromoted a significant increase in the RID when the same juvenilewas re-exposed 30 min after the first encounter, indicating a dis-ruption in the short-term social recognition ability of aging micecaused by i.n. MPTP infusion. Repeated treatment with agmatineprevented the deficit in social recognition ability induced by i.n.MPTP administration, causing a significant reduction in the RIDwhen the familiar juvenile was re-exposed after 30 min (Fig. 3).

3.3. Effects of agmatine on the general neurological state of agingmice infused intranasally with MPTP

The general neurological state of the animals was evaluatedby the NSS at 13 days after i.n. MPTP administration. As illus-trated in Fig. 4, Kruskal–Wallis non-parametric test followed byDunn’s post hoc tests indicated that mice from the control/MPTPgroup scored significantly higher [P ≤ 0.05] than the control/controlgroup. Of high importance, this increase in NSS score induced byi.n. MPTP administration was prevented by the pretreatment with

Fig. 4. Effects of the pretreatment (during 5 consecutive days) with control(i.p.) or agmatine (30 mg/kg, i.p.) on the general neurological state of 15-month-old female C57BL/6 mice evaluated in the Neurological Severity Score 13 daysafter i.n. infusion of control or MPTP (1 mg/nostril). The results are shown asmedian (interquartile ranges) of NSS points [control/control (n = 9); control/MPTP(n = 8); agmatine/control (n = 10); and agmatine/MPTP (n = 9)]. *P ≤ 0.05 comparedto the control/control group. #P ≤ 0.05 compared to the control/MPTP group(Kruskal–Wallis non-parametric test followed by Dunn’s multiple comparisontests).

F.C. Matheus et al. / Behavioural Brain Research 235 (2012) 263– 272 267

Fig. 5. Effects of the pretreatment (during 5 consecutive days) with control (i.p.) or agmatine (30 mg/kg, i.p.) on the spontaneous locomotor activity of 15-month-old femaleC57BL/6 mice evaluated during 5 min in the activity chambers and open field at 3 and 19 days, respectively, after i.n. infusion of control or MPTP (1 mg/nostril). Data areexpressed as the mean ± S.E.M. of the total distance traveled (A) and average speed (B) in the activity chambers [control/control (n = 10); control/MPTP (n = 9); agmatine/control(n = 10); and agmatine/MPTP (n = 9)]; and number of crossings (C) and rearing (D) in the open field [control/control (n = 9); control/MPTP (n = 7); agmatine/control (n = 10);and agmatine/MPTP (n = 9)]. *P ≤ 0.05 compared to the control/control group. #P ≤ 0.05 compared to the control/MPTP group (two-way ANOVA followed by Newman–Keulstest).

agmatine (30 mg/kg, i.p.), suggesting a protective effect of agmatineagainst the neurological impairments induced by MPTP in agingmice (Fig. 4).

3.4. Effects of agmatine on the spontaneous locomotor activity ofaging mice infused intranasally with MPTP

The results of locomotor activity of aging mice evaluated for5 min in the activity chambers and open field arena at 3 and 19 days,respectively, after i.n. MPTP (1 mg/nostril) administration are sum-marized in Fig. 5. Two-way ANOVA revealed no significant effectsfor the main factors and their interaction in the total distance trav-eled [pretreatment: F1,33 = 1.21; P = 0.28; treatment: F1,33 = 0.45;P = 0.48; interaction: F1,33 = 0.28; P = 0.60] (Fig. 5A) and the averagespeed [pretreatment: F1,33 = 1.31; P = 0.29; treatment: F1,33 = 0.28;P = 0.60; interaction: F1,33 = 0.32; P = 0.60] (Fig. 5B) evaluated in theactivity chambers.

On the other hand, two-way ANOVA revealed significant effectsfor the treatment factor [F1,29 = 5.86; P ≤ 0.05] and the interactionbetween pretreatment and treatment [F1,29 = 4.26; P ≤ 0.05], but notfor the pretreatment factor [F1,29 = 1.26; P = 0.27], in the numberof squares crossed in the open field. Post hoc comparisons indi-cated a significant reduction in the number of crossings in theMPTP/control group that was not observed in the agmatine/MPTPgroup (Fig. 5C).

Regarding the number of rearings, two-way ANOVA revealedsignificant effects for the main factors [pretreatment: F1,29 = 4.23;P ≤ 0.05; treatment: F1,29 = 6.70; P ≤ 0.05] and their inter-action [F1,29 = 4,23; P ≤ 0.05] in this parameter. SubsequentNewman–Keuls test indicated that the reduction in the number of

rearings induced by i.n. MPTP administration was prevented by thepretreatment with agmatine (Fig. 5D). Therefore, the locomotoractivity of aging mice was only disrupted at later periods after i.n.MPTP administration which was prevented by pretreatment withagmatine (30 mg/kg, i.p.).

3.5. Agmatine prevents the loss of dopaminergic neurons inducedby i.n. MPTP administration in aging mice

With the purpose of determining the relationship between themotor impairments observed in the open field at later periods afteri.n. MPTP administration in aging mice and dopaminergic cell deathin the nigrostriatal pathway, the evaluation for TH-positive cells inthe substantia nigra was performed 21 days after i.n. administrationof MPTP by immunohistochemistry. Fig. 6A–D shows representa-tive photomicrographs of TH immunohistochemistry in the ventralmesencephalon containing SNpc.

As can be seen in Fig. 6E, two-way ANOVA revealed signif-icant effects for the treatment factor [F1,16 = 8.73; P ≤ 0.01] andthe interaction between pretreatment and treatment [F1,16 = 4.52;P ≤ 0.05], but not for the pretreatment factor [F1,16 = 0.52; P = 0.48],in the number of TH-positive cells in the SNpc. SubsequentNewman–Keuls tests showed that the i.n. administration of MPTPinduced a pronounced reduction (about 50%) of TH immunostain-ing in the SNpc of aging mice when compared to the control/controlgroup (P ≤ 0.05). Of high importance, the pretreatment with agma-tine (30 mg/kg, i.p.) was able to attenuate the loss of TH-positiveneurons in the SNpc of MPTP-treated mice when compared to con-trol/control group (Fig. 6E).

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Fig. 6. Effects of the pretreatment (during 5 consecutive days) with control (i.p.) or agmatine (30 mg/kg, i.p.) on tyrosine hydroxylase (TH)-positive cells in the substantianigra pars compacta (SNpc) of 15-month-old female C57BL/6 mice evaluated through immunohistochemistry at 21 days after i.n. infusion of control or MPTP (1 mg/nostril).(A–D) Representative images of TH immunostaining in the ventral mesencephalon containing SNpc of animals (Scale bar = 200 !m). (E) Relative quantification of the numberof TH-positive neurons per mm2 in SNpc of mice. Values represent the mean ± SEM (n = 5 animals per group). *P ≤ 0.05 compared to the control/control group (two-wayANOVA followed by Newman–Keuls test).

3.6. Agmatine prevents decreased hippocampal glutamate uptakeinduced by i.n. MPTP administration in aging mice

Glutamate clearance from extracellular space is an importantmechanism related to the reduction of glutamate excitotoxicity.With the purpose of determining possible alterations in gluta-matergic neurotransmission following MPTP treatment, glutamate

uptake was measured in the hippocampus of aging mice 21 daysafter i.n. MPTP administration.

As illustrated in Fig. 7, the i.n. MPTP administration sig-nificantly decrease [F1,14 = 5.16; P ≤ 0.05] the glutamate uptakeinto the hippocampus of aging mice. The pretreatment withagmatine did not alter per se the basal hippocampal glutamateuptake [F1,14 = 2.62; P = 0.70], but it prevented the MPTP-induced

F.C. Matheus et al. / Behavioural Brain Research 235 (2012) 263– 272 269

Fig. 7. Effects of the pretreatment (during 5 consecutive days) with control (i.p.)or agmatine (30 mg/kg, i.p.) on hippocampal l-[3H]glutamate uptake in 15-month-old female C57BL/6 mice infused intranasally with control or MPTP (1 mg/nostril). Atthe 21th day after i.n. MPTP administration, mice were sacrificed and the hippocam-pal slices processed for glutamate uptake assay in vitro as described in Section 2.Values are expressed as mean ± SEM [control/control (n = 4); control/MPTP (n = 3);agmatine/control (n = 5); and agmatine/MPTP (n = 4)]. P ≤ 0.05 compared to the con-trol/control group (two-way ANOVA followed by Newman–Keuls test).

reduction of glutamate uptake [interaction factor: F1,14 = 4.88;P ≤ 0.05] (Fig. 7).

3.7. Effects of agmatine on the monoamine oxidase activity

As can be seen in Fig. 8, Student’s t-tests indicated that thecurrent tested concentrations of agmatine (0.01–1000 !m) hadno significant effect on either MAO-A [t = 1.30, P = 0.25] or MAO-B [t = 0.96, P = 0.38] activities in the mouse brain mitochondrialhomogenates.

4. Discussion

Therapeutic strategies that slow or stop the neurodegenerativeprocess of PD are expected to have a major impact on the treatmentof this disease [47]. The current hypothesis about the mechanismsby which neurons come into necrotic or apoptotic process of degen-eration has led to belief that the use of drugs modulating thefunction of glutamate NMDA receptors may have beneficial effectsin PD therapy [10,63]. In this context, there is increasing evidenceof the neuprotective effects of agmatine, which among other possi-ble targets blockades NMDA receptors, against different insults of

Fig. 8. Effects of agmatine on mouse brain mitochondrial MAO-A and MAO-B activ-ities in vitro. Agmatine was tested in a concentration range of 0.01–1000 !m. Thevalues represent the mean ± S.E.M. of three individuals experiments, performed induplicate.

the CNS including the dopaminergic neurodegeneration induced byPD-mimetic toxins such as MPTP [30] and rotenone [15,16].

The current data corroborates the neuroprotective potentialof agmatine in PD since it attenuated the dopaminergic cellloss in the SNpc of aging mice infused intranasally with MPTP(1 mg/nostril). Moreover, the present study provides the firstpreclinical data demonstrating that repeated treatment with agma-tine (30 mg/kg, i.p.) improves short-term memory and motorimpairments displayed by MPTP-treated aging mice. Finally, theobserved behavioral benefits of exogenous agmatine treatmentwere accompanied by the prevention of MPTP-induced decreaseof hippocampal glutamate uptake.

Recent studies performed by our group and others with theadministration of exogenous agmatine in laboratory animals haveidentified several relevant functions of this substance that are ofpotential therapeutic importance, including anticonvulsant [17],antinociceptive [28,54,64], anxiolytic [32] and antidepressant-like[73,74] actions. Moreover, age-related changes in agmatine levelsin various brain structures have been demonstrated, thus indicatingthe potential involvement of agmatine in aging process [45].

In the present study, the repeated treatment with agmatine(30 mg/kg, i.p.) during 5 consecutive days was able to attenuatesignificantly the mortality of MPTP-treated aging mice. The acutetoxicity effects of MPTP are largely attributed to peripheral mecha-nisms [40]. For instance, MPTP and its toxic metabolite MPP+ havebeen shown to have a variety of peripheral effects including cardiacnoradrenaline depletion [2], adrenal noradrenaline and dopaminerelease [2], hypothermia [27] and neuromuscular blockade viabinding to nicotinic acetylcholine receptors [39]. It must be con-ceded that, at present stage, it not possible to determine the exactsite of action and molecular mechanisms underlying agmatineattenuated the mortality of MPTP-infused aging mice. Neverthe-less, based on previous literature demonstrating that agmatineinhibits sympathetically-induced tachycardic responses [14] andnoradrealine release [60], a speculative hypothesis is that agmatinemay prevent the alterations in the cardiovascular system verifiedfollowing MPTP administration.

Regarding PD symptoms, an increasing number of studies havedemonstrated that PD seems to be a multidimensional disease, andbesides motor deficits, it is associated with a number of senso-rial, cognitive and emotional disturbances that result in a loss inquality of life of the individuals [13]. In this context, in a recentseries of studies we demonstrated that a single i.n. infusion of MPTPin rodents produces diverse signs of PD such as impairments inolfactory, cognitive, emotional and motor functions [12,50,57–59].Moreover, the i.n. MPTP administration seems to affect the rodents’brain in a region- and time-dependent manner (for review see[57]). For instance, we have observed increased susceptibility ofolfactory bulb to i.n. MPTP toxicity, with a marked reduction inTH-positive neurons and dopamine depletion occurring 24 h afteri.n. MPTP infusion. On the other hand, these alterations were onlyobserved later (14–21 days after intransal MPTP administration) inthe SNpc and striatum. Therefore, the existence of compensatorymechanisms, such as the increase in the number of TH-positivestriatal cells and a downregulation of dopamine uptake in surviv-ing dopaminergic fibers in the striatum [8], may be responsible forthe lack of striatal changes until 14 days post-MPTP. Consistentwith these observations, the current findings indicated no signifi-cant alterations in the total distance traveled and the average speedby aging mice in the activity chambers at 3 days after i.n. MPTPadministration.

Since the i.n. administration of MPTP does not cause, at least atinitial periods, gross motor alterations that would preclude assess-ment of cognitive functions, we investigated the impact of i.n.MPTP administration on social recognition memory of aging mice.Aging mice infused with MPTP (1 mg/nostril) spent significantly

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more time investigating the juvenile during the second presenta-tion than they did in the first encounter, suggesting an impairedability to recognize the juvenile mouse after a short time. Previ-ous studies from our group have demonstrated that rodents treatedintranasally with MPTP performed normally in the long-term reten-tion session (24 h after training) of the inhibitory avoidance task[12,58] and in the spatial reference memory version of the watermaze [59]. In contrast, MPTP-infused animals displayed a poor per-formance in the short-term retention session (1.5 h after training)of the inhibitory avoidance task [12,58] as well as in the workingmemory version of the water maze [59]. These findings are consis-tent with the view of human studies suggesting that PD patientspresent cognitive deficits mainly in working memory and short-term memory tasks with long-term spatial (declarative) memoriesmostly spared [11].

Of high importance, the present findings demonstrate that thepretreatment with agmatine (30 mg/kg, i.p.) during 5 consecutivedays was able to prevent the short-term social memory deficitsof MPTP-treated aging mice. Therefore, from these limited resultsit appears that agmatine might be particularly useful to restorememory processes in PD. The current results are in accord withprevious findings indicating the cognitive-enhancing properties ofagmatine in diverse animal models of brain injury. For instance, therepeated administration of agmatine (5–40 mg/kg, i.p.) preventedlearning and memory impairments in rodents induced by infusionof aggregated beta-amyloid(25-35) peptide [7], lipopolysaccha-ride [71], streptozotocin [9] and scopolamine [49]. Moreover,recent studies [44,45,67] have demonstrated that spatial learningin rodents induces elevation in agmatine levels at synapses in therat hippocampus, providing further evidence of its participation inlearning and memory processes.

Additionally, in the present study we observed a later (19 daysafter i.n. MPTP administration) reduction in the locomotor activ-ity of MPTP-treated 15-month-old female C57BL/6 mice evaluatedin the open field that was accompanied by a marked reduction(about 50%) of TH-positive neurons in the SNpc. These findingscontrast with our previous study demonstrating that young adult(6-months-old) male C57BL/6 mice infused intranasally with thesame dose of MPTP do not present gross motor alterations [58].Therefore, the current data indicates that the age and gender ofthe animals represent important factors that modulate the appear-ance of motor symptoms in the i.n. MPTP model of PD. Reinforcingthe current findings, previous studies have demonstrated thatdopamine-depleting effects and motor impairments induced byi.p. MPTP administration in mice are age- [41,52] and gender-dependent [3,26,41,53,68].

Epidemiological studies have shown a prevalence of PD in mencompared to women [20]. In women, the age at onset of PD cor-relates with the end of the fertile life [62]. However, results fromprevious preclinical studies addressing gender-related differenceson MPTP toxicity have been inconsistent, with increased suscep-tibility to MPTP-induced behavioral and neurochemical changesbeen described for both male [3,26] and female [41,53,68] mice.Interestingly, Unzeta et al. [68] observed significant differences inMAO-A and MAO-B activities during the oestrous cycle as well asbetween adult male and female mice. Since the neurotoxic effectsof MPTP depend on its conversion to the MPP+ by MAO-B, theobserved differences in MAO-B activity may be involved in thegender-related effects of MPTP. Therefore, the use of female micewith 15-months-old in the present study, which at this age showcessation of estrous cycle with very low levels of estrogen [24],attenuates the hormonal variability which could interfere withMPTP toxicity.

Of high importance, the administration of agmatine demon-strated once again its neuroprotective properties as previouslydescribed in several models of neuronal damage [31,42,72],

preventing the locomotor impairments in the open field and thedecrease of the TH immunoreactivity in the SNpc induced by i.n.infusion of MPTP in aging mice. These results corroborate recentfindings on agmatine neuroprotection in cellular models of PD-likeneurodegeneration [15,16]. Moreover, Gilad et al. [30] publisheda pioneer study demonstrating that the treatment with agmatine(100 mg/kg, i.p.) during 5 days attenuated MPTP-induced reductionof synaptosomal dopamine uptake when administered 8 h afterMPTP injection (40 mg/kg, i.p., for 2 days). Taken together, theseresults suggest that agmatine may represent a potential disease-modifying therapy for PD.

It is well known that MAO-B inhibition reduces the generationof the MPP+ from MPTP, protecting against the dopaminergic celldeath in the SNpc [36]. In accordance with previous literature [30],we observed that agmatine does not interfere with MAO-B activityon mouse brain mitochondrial homogenates, indicating that MAO-B-catalyzed conversion of MPTP to MPP+ is not affected. However,the evaluation of the time-course of MPP+ kinetic in the mousebrain after i.n. MPTP administration constitutes a very interestingfield that requires additional research.

The neuroprotective effects of agmatine may result from dif-ferent mechanisms including blocking of NMDA receptors [70],inhibition of nitric oxide synthase (NOS) [29], oxygen radicalscavenging [6] and protection against mitochondrial membranepotential collapse [4–6]. However, the sequence of events lead-ing to the protective effects of agmatine against cell damage hasnot been fully elucidated. Here we observed that agmatine wasable to prevent the decrease of hippocampal glutamate uptake inaging mice following i.n. MPTP administration. Corroborating ourfindings, previous studies [18,35] have demonstrated that MPTPdecreases glutamate uptake by astrocytes in cell culture. Therefore,one possible mechanism by which agmatine may exert protectiveeffects against MPTP neurotoxicity may be due to the modulation ofglutamate reuptake into neural cells, the main mechanism respon-sible for decreasing extracellular glutamate levels, thus attenuatingglutamate neurotoxicity.

5. Conclusions

The present findings reinforce the i.n. MPTP administration as avaluable rodent model for testing novel palliative and neuroprotec-tive compounds for PD and demonstrate that the age of the animalsrepresent an important factor that modulate the appearance ofmotor symptoms in this model. More importantly, the presentstudy provides the first preclinical data indicating that repeatedsystemic treatment with agmatine prevents short-term memoryand motor impairments as well as dopaminergic cell loss in theSNpc of aging mice submitted to an experimental model of PD.These results provide new insights in experimental models of PD,indicating that agmatine may represent a new therapeutic tool forthe management of cognitive and motor symptoms of PD, togetherwith its neuroprotective potential.

Acknowledgements

This work was supported by grants from Conselho Nacionalde Desenvolvimento Científico e Tecnológico (CNPq), Coordenac ãode Aperfeic oamento de Pessoal de Nível Superior (CAPES), Pro-grama de Apoio aos Núcleos de Excelência (PRONEX – ProjectNENASC), Fundac ão de Apoio à Pesquisa do Estado de Santa Cata-rina (FAPESC), FINEP (Financiadora de Estudos e Projetos-IBN-Net#01.06.0842-00) and INCT (Instituto Nacional de Ciência e Tec-nologia) for Excitotoxicity and Neuroprotection. FCM and AACreceive scholarships from CNPq. CPF, JF, RW, ARSS, CIT and RDP are

F.C. Matheus et al. / Behavioural Brain Research 235 (2012) 263– 272 271

supported by research fellowships from CNPq. The authors have nofinancial or personal conflicts of interest related to this work.

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