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Behavioural Research 235 (2012) 263–272

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Behavioural Brain Research

j ournal homepage: www.elsevier.com/locate/bbr

Research report

Neuroprotective effects of in mice infused with a single intranasal

administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

a a,b b c c

Filipe C. Matheus , Aderbal S. Aguiar Jr. , Adalberto A. Castro , Jardel G. Villarinho , Juliano Ferreira ,

d e f b a,

Cláudia P. Figueiredo , Roger Walz , Adair R.S. Santos , Carla I. Tasca , Rui D.S. Prediger ∗

a

Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, UFSC, 88049-900 Florianópolis, SC, Brazil

b

Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, UFSC, 88040-900 Florianópolis, SC, Brazil

c

Departamento de Química, Universidade Federal de Santa Maria, UFSM, Santa Maria, RS 97105-900, Brazil

d

Departamento de Fármacos, Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, UFRJ, Rio de Janeiro, RJ, Brazil

e

Departamento de Clínica Médica, Hospital Universitário, Universidade Federal de Santa Catarina, UFSC, Florianópolis, SC, Brazil

f

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 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 a b s t r a c t

Article history: We have recently demonstrated that rodents treated intranasally with 1-methyl-4-phenyl-1,2,3,6-

Received 24 May 2012

tetrahydropyridine (MPTP) suffered impairments in olfactory, cognitive, emotional and motor functions

Received in revised form 9 August 2012

associated with time-dependent disruption of dopaminergic in different brain struc-

Accepted 12 August 2012

tures conceivably analogous to those observed during different stages of Parkinson’s disease (PD).

Available online 17 August 2012

Agmatine, an endogenous metabolite, has been proposed as a novel neuromodulator that plays

protective roles in several models of neuronal cellular damage. In the present study we demonstrated

Keywords:

that repeated treatment with agmatine (30 mg/kg, i.p.) during 5 consecutive days increased the sur-

Agmatine

vival rate (from 40% to 80%) of 15-month-old C57BL/6 female mice infused with a single intranasal (i.n.)

Parkinson’s disease

1-Methyl-4-phenyl-1,2,3,6 administration of MPTP (1 mg/nostril), improving the general neurological status of the surviving animals.

tetrahydropyridine (MPTP) Moreover, pretreatment with agmatine was found to attenuate short-term social memory and locomo-

Intranasal tor activity impairments observed at different periods after i.n. MPTP administration. These behavioral

Aging mice benefits of exogenous agmatine administration were accompanied by a protection against the MPTP-

Non-motor symptoms

induced decrease of hippocampal glutamate uptake and loss of dopaminergic in the substantia

nigra pars compacta of aging mice, without altering brain B (MAO-B) activity. These

results 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 its

neuroprotective effects. © 2012 Elsevier B.V. All rights reserved.

1. Introduction

Parkinson’s disease (PD) is a debilitating disease primarily

characterized by the progressive loss of neuromelanin-containing

dopaminergic neurons in the pars compacta (SNpc)

with presence of eosinophillic, intracytoplasmic, proteinaceous

Corresponding author at: Departamento de Farmacologia, Universidade Federal

∗ inclusions termed as Lewy bodies and dystrophic Lewy neurites

de Santa Catarina, Campus Trindade, 88049-900 Florianópolis, SC, Brazil.

in surviving neurons [37]. At the time of diagnosis, patients typi-

Tel.: +55 48 3721 9491; fax: +55 48 3337 5479.

E-mail address: [email protected] (R.D.S. Prediger). cally display an array of motor impairments including bradykinesia,

0166-4328/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2012.08.017

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

resting tremor, rigidity, and postural instability. Although most of It has been well established that aging is the most prominent risk

the typical motor impairments are due to the loss of nigrostriatal factor for PD. However, preclinical studies addressing the behav-

dopaminergic neurons, PD affects multiple neuronal systems both ioral and effects of dopaminergic such

centrally and peripherally, leading to a constellation of non-motor as MPTP have been widely performed in young adult animals which

symptoms including olfactory deficits, affective disorders, memory may represent one possible explanation for the limited success

impairments, as well as autonomic and digestive dysfunction [13]. on translational research in PD. Therefore, the aim of the present

These non-motor features of PD do not meaningfully respond to study was to evaluate the potential of the repeated administration

dopaminergic medication and are a challenge to the clinical man- of exogenous agmatine to prevent behavioral and neurochemical

agement of PD [13]. changes induced by a single i.n. MPTP administration in aging mice.

Numerous epidemiological and experimental studies suggest

that exposure to agricultural chemicals, viruses, metals, and other 2. Materials and methods

toxins contribute to its pathogenesis (for review see [19,56]).

2.1. Animals

In some cases such agents conceivably enter the brain via the

olfactory neuroepithelium, a concept termed the olfactory vector Experiments were conducted using 15-month-old female C57BL/6 mice weigh-

hypothesis [22,56]. In this context, we have recently proposed a 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 per

new experimental model of PD consisting of a single intranasal

cage) and maintained in a room under controlled temperature (23 1 ◦C) and 12 h

(i.n.) administration of the proneurotoxin 1-methyl-4-phenyl- ±

light cycle (lights on 7:00 AM), with free access to food and . The animals were

1,2,3,6-tetrahydropyridine (MPTP) in rodents [12,50,57–59]. Young

treated, manipulated and euthanized according to the “Principles of Laboratory Ani-

adult Wistar rats and C57BL/6 mice (3–6-months-old) treated mal Care” (NIH publication no. 80-23, revised 1996) and approved by the Committee

intranasally with MPTP suffer impairments in olfactory, cognitive, on 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 were

emotional and motor functions conceivably analogous to those

made to minimize the number of animals used and their suffering.

observed during different stages of PD. Such infusion causes time-

dependent loss of hydroxylase (TH) in the olfactory bulb

2.2. Drugs and treatment

and SNpc, resulting in significant depletion in differ-

ent brain areas [57–59]. We have also identified some pathogenic The animals were allocated to the following groups: (i) control + control (n = 13),

mechanisms possibly involved in the neurodegeneration induced (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-

by i.n. administration of MPTP including mitochondrial dysfunc-

anced across the four groups. Agmatine sulfate (Sigma Chemicals Co., USA) was

tion, oxidative stress, activation of apoptotic cell death mechanisms

freshly dissolved in 0.9% NaCl (saline) to a final concentration of 3 mg/ml before

and excitotoxicity (for review see [57]). Therefore, each daily treatment. Animals were administered by i.p. route once daily for 5 con-

the i.n. MPTP administration seems to represent a valuable rodent secutive days with control solution (saline) or agmatine (30 mg/kg) in a volume of

0.1 ml/10 g of body weight. The dose of agmatine utilized was chosen based on previ-

model for testing novel drugs for both motor and non-motor symp-

ous studies conducted in our laboratory [28,54,64]. One hour after the last

toms relief as well as the discovery of compounds to modify the

of agmatine, the animals were infused intranasally with a single bilateral dose of

course of PD.

MPTP (1 mg/nostril) or control solution (saline) (Fig. 1).

On the other hand, there is increasing evidence that alter- MPTP HCl (Sigma Chemicals Co., USA) was administered by i.n. route according

ations in glutamatergic neurotransmission have a pivotal role in to the procedure previously described [21] and modified in our laboratory [58].

Briefly, mice were lightly anaesthetized with isoflurane 0.96% (0.75 CAM; Abbot

the pathophysiology of PD [10,63]. For instance, dopaminergic

Laboratórios do Brasil Ltda., RJ, Brazil) using a vaporizer system (SurgiVet Inc., WI,

neurons in the SNpc receive moderate excitatory glutamater-

USA) and a 7 mm piece of PE-10 tubing was inserted through the nostrils. The tubing

gic neurons input from the subthalamic nucleus (STN) [33]. It is was connected to a peristaltic pump set at a flow rate of 12.5 ␮l/min. The MPTP HCl

postulated that overstimulation of glutamate receptors on nigral was dissolved in saline at a concentration of 20 mg/ml, after which it was infused for

4 min (1 mg/nostril). The control solution consisted of saline. Animals were given a

dopaminergic neurons may be involved in the neuronal degenera-

1 min interval to regain normal respiratory function and then this procedure was

tion and progression of PD [10,63]. Moreover, it was demonstrated

repeated with infusions administered through the contralateral nostrils.

a pronounced increase in the extracellular levels of glutamate

in the substantia nigra of mice treated chronically with MPTP

2.3. Survival analysis

[48]. Indeed, recent studies have demonstrated that d-,

a partial of the binding site of the N-methyl-d- 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.

aspartate (NMDA) , improves mnemonic impairments and

treatment with agmatine or control (data not shown). The number of deaths was

anxiety-like behaviors observed in MPTP-lesioned monkeys [65]

monitoring until the 21st day after the i.n. MPTP administration for later assembly

and rats [38,69]. Finally, there are promising findings from clinical

of the survival curve.

[1,23] and preclinical studies of the efficacy of the NMDA recep-

tor antagonist for the treatment of PD [43]. Thus, drugs 2.4. Behavioral tests

modulating the function of glutamate NMDA receptors may have

During a period of 3–19 days after the i.n. administration of MPTP, the animals

beneficial effects in PD therapy.

were submitted to a battery of behavioral paradigms that included the activity cham-

In this context emerges agmatine, a polyamine that is synthe-

ber, social recognition, neurological severity score and open-field tasks (Fig. 1). The

sized after decarboxylation of l-arginine by arginine decarboxylase

time point for the performance of each behavioral task was chosen based on previ-

and it is hydrolyzed to by and that ous studies using the i.n. MPTP model [12,50,57]. All tests were carried out between

has been recently proposed as neuromodulator [34,61]. In the brain, 9:00 and 14:00 h and they were scored by the same rater in an observation room

where the mice had been habituated for at least 1 h before the beginning of the tests.

agmatine is stored in synaptic vesicles [34] and is released by

2+ Behavior was monitored through a video camera positioned above the apparatuses

Ca -dependent depolarization [61] with multiple molecular tar-

and the images were later analyzed with the ANY Maze video tracking (Stoelting

gets proposed, including the binding and blockage of glutamate Co., Wood Dale, IL, USA) by an experienced experimenter who was unaware of the

NMDA receptors [70]. Age-related changes in agmatine levels in experimental group of the animals tested.

various brain structures have been described indicating potential

involvement of agmatine in aging process [45]. Of high impor- 2.4.1. Activity chambers

In order to assess early effects of MPTP on locomotor activity, the animals were

tance, recent studies have demonstrated the protective effects

tested 3 days after i.n. MPTP administration in activity chambers. The chambers were

of exogenous agmatine administration in animal models of neu-

made of black fiberglass (50 cm 25 cm 15 cm) and the experiments were per-

× ×

rodegenerative disorders such as PD [15,16,30] and Alzheimer’s formed in a sound-attenuated room under low-intensity light (12 lux). Each mouse

disease [7]. was 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 a

single 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 with Technology, USA) as described previously [58]. Following quenching of endogenous

10% between animals. peroxidase with 3% peroxide in for 20 min, high tempera-

ture antigen retrieval was performed by immersion of the slides in a water bath

2.4.2. Social recognition at 95–98 ◦C in 10 mM trisodium citrate buffer pH 6.0, for 45 min. After overnight

Short-term social memory was assessed 7 days after i.n. MPTP administration incubation at 4 ◦C with primary , the slides were washed with PBS and

with the social recognition task previously evaluated in our laboratory [58]. Juve- incubated with the appropriate biotinylated secondary , and then pro-

nile female C57BL/6 mice (25–30 days old) served as social stimuli for the adult cessed using the Vectastain Elite ABC reagent (Vector Laboratories, Burlingame, CA,

mice in the social recognition task. The adult animals were isolated in individual USA) according to the manufacturer’s instructions. The sections were washed in PBS,

cages during seven days before the task. All juveniles were isolated in individual and the visualization was completed by using 3,3-diaminobenzidine (DAB) (Dako

cages for 20 min prior to the beginning of the experiment. The social recognition Cytomation) in chromogen solution and counterstained with Harris’s hematoxylin.

task consisted of two successive presentations (5 min each), separated by an inter- Tissues from the four groups were placed on the same slide and processed under the

trial interval of 30 min, where the juvenile was placed in the home cage of the adult same conditions. We included negative control that consisted in replace the primary

mouse and the time spent by the adult in investigating the juvenile (nosing, sniffing, antibody by nonimmune serum in equivalent concentration.

grooming, or pawing) was recorded in the two presentations. Time spent in social The number of TH-stained positive cells in the SNpc was assessed at three levels

investigation by the adult mouse was measured and then expressed for each ani- between coordinates 2.75 mm and 2.92 mm with respect to the bregma. Three

− −

mal as the ratio of the second exposure to the first exposure (Ratio of Investigation alternate 4 ␮m paraffin sections with an individual distance of 60 ␮m of each sec-

Duration (RID)). A reduction in RID reflects a decrease in investigation behavior dur- tion were obtained, and the number of TH-stained positive cells was determined

ing the second encounter, demonstrating the recognition ability of the adult mouse. upon visual inspection at the SNpc with optical microscope (Eclipse 50I; Nikon,

This transformation was chosen in order to minimize day-to-day variations on the Melville, NY) by using a counting grid at 400 magnification. Results were expressed

× 2

baseline of performance and to equalize variances among different groups [12]. as mean number of TH-stained positive cells per mm from three sections.

3

2.4.3. Neurological severity score 2.6. l-[ H]glutamate uptake

Thirteen days after i.n. administration of MPTP, mice performed the 10-point

neurological severity score (NSS), a composite behavioral scale designed to measure The glutamate uptake assay was evaluated as previously described [55]. The

the general neurological state previously described [25] and recently evaluated in mice were decapitated 21 days after i.n. MPTP administration and the hippocampi

our laboratory [66]. Mice were assessed for the following items: presence of paresis, were quickly removed and stored in KRB (in mM = 122 NaCl, 3 KCl, 1.3 CaCl2, 1.2

inability to walk straight, impairment of seeking behavior, absence of perceptible MgSO4, 0.4 KH2PO4, 25 NaHCO3 and 10 d-) previously aerated with carbogen

startle reflex, inability to exit a 30 cm diameter circle, inability to walk on 3, 2, and (95% O2–5% CO2) to reach pH 7.4. The tissue sections (400 ␮m thick) were obtained

1 cm wide beams, and inability to balance on a 0.7 cm-wide beam and a 0.5 cm- using a tissue slicer (Mcilwain Tissue Chopper, Australia). On average, five slices from

diameter round beam for at least 10 s. If mice showed the impairment described by the middle of the were obtained and placed in a 96-well multiwell

an item, a value of 1 was added to total NSS score. Higher scores in the NSS indicate plate containing KRB, pH 7.4. The slices were incubated for 1 h before the gluta-

greater neurological impairment [66]. mate uptake assays were performed. After incubation, the hippocampal slices were

washed for 15 min at 37 ◦C in a Hank’s balanced salt solution (HBSS), composition in

2.4.4. Open field mM: 1.29 CaCl2, 136.9 NaCl, 5.36 KCl, 0.65 MgSO4, 0.27 Na2HPO4, 1.1 KH2PO4, and 5

3

The spontaneous locomotor activity of the animals was evaluated in an open- HEPES. Uptake was assessed using 0.33 ␮Ci/ml l-[ H]glutamate with 100 ␮m unla-

field arena at 19 days after i.n. MPTP administration. The apparatus, made of wood beled glutamate in a final volume of 300 ␮l. Incubation was immediately stopped

covered with impermeable Formica, had a black floor of 50 cm 50 cm (divided after 7 min by discarding the incubation medium and the slices were submitted to

×

by white lines into 9 squares of equal size) and transparent walls, 40 cm high. Each two ice-cold washes with 1 ml of HBSS. The slices were solubilized by adding a solu-

mouse was placed in the center of the open field and the numbers of squares crossed tion with 0.1% NaOH/0.01% SDS and incubated overnight. Aliquots of slice lysates

3

and rearing were registered during 5 min. The apparatus was cleaned with ethanol were taken for determination of the intracellular content of l-[ H]glutamate by scin-

solution (10% v/v) and dried with paper towels after each trial in order to avoid odor tillation counting. -independent uptake was determined by using

impregnation. instead of in the HBSS. Unspecific sodium-independent

uptake was subtracted from total uptake to obtain the specific sodium-dependent

3

glutamate uptake. Results were expressed as nmol of l-[ H]glutamate taken up per

2.5. Immunohistochemistry for (TH)

milligram of per minute.

For the investigation of possible neuroprotective effects of agmatine against the

loss of dopaminergic neurons induced by i.n. MPTP administration, five animals of 2.7. Monoamine oxidase (MAO) assay

each group were intracardially perfused with 4% paraformaldehyde in physiologi-

cal saline (NaCl 0.9%) at 21 days after MPTP treatment. were collected and To investigate whether the treatment with agmatine may interfere with the gen-

+

fixed in a phosphate buffered saline (PBS) solution containing 4% paraformalde- eration of the toxic metabolite 1-methyl-4-phenylpyridinium (MPP ) from MPTP,

hyde for 24 h at room temperature, dehydrated by graded ethanol, and embedded agmatine was tested for its in vitro inhibitory potential on mouse MAO-A and MAO-

in paraffin. Immunoreactivity of TH-positive neurons in the substantia nigra pars B activities in brain mitochondrial homogenates by a fluorometric method using

compacta (SNpc) was assessed on paraffin tissue sections (4 ␮m), using the anti-TH kynuramine as substrate, as previously described [46]. Brain (all regions without

monoclonal antibody (1:300, catalog MAB318, Millipore/Chemicon International, ) 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 mice

infused intranasally with control or MPTP (1 mg/nostril). The lines represent the

Fig. 3. Effects of the pretreatment (during 5 consecutive days) with control (i.p.) or

percentage survival animals of each group over the course of 21 days after the

agmatine (30 mg/kg, i.p.) on the social recognition memory of 15-month-old female

i.n. MPTP administration. * P < 0.05 compared to the percentage of survival of the

C57BL/6 mice evaluated in the social recognition task 7 days after i.n. infusion of

control/control group. # P < 0.05 compared to the percentage of survival of the

MPTP (1 mg/nostril). Data are expressed as the mean S.E.M. of RIDs (i.e. the ratio

agmatine/MPTP group (Breslow–Gehan–Wilcoxon test). ±

of the second exposure to the first exposure) when the same juvenile was exposed

for 5 min with an interval of 30 min [control/control (n = 10); control/MPTP (n = 9);

according to the method of Naoi et al. [51]. The obtained mitochondrial pellet was agmatine/control (n = 10); and agmatine/MPTP (n = 9)]. *P 0.05 compared to the

suspended in 10 mM sodium phosphate buffer (pH 7.4) to 100–300 mg/mL and control/control group. #P 0.05 compared to the control/MPTP group (two-way

then was used for assay. Briefly, assays were performed in duplicate in a final ANOVA followed by Newman–Keuls test).

volume 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 by

P 0.001] and their interaction [F1,33 = 8,44, P 0.01] in the ratio of

incorporating 250 nM selegiline (selective MAO-B inhibitor) or 250 nM clorgyline ≤ ≤

investigation duration (RID).

(selective MAO-A inhibitor) into the reaction mixture. The reaction mixture (con-

taining mitochondrial fractions, agmatine and inhibitors) was pre-incubated at 37 ◦C Post hoc comparisons indicated that the i.n. MPTP treatment

for 5 min and the reaction was started by addition of 50 ␮l of kynuramine (90 ␮m promoted a significant increase in the RID when the same juvenile

for MAO-A and 60 ␮m for MAO-B). Agmatine was tested in a concentration range

was re-exposed 30 min after the first encounter, indicating a dis-

of 0.01–1000 ␮m and the results were expressed as percentage of control (tube

ruption in the short-term social recognition ability of aging mice

without agmatine).

caused by i.n. MPTP infusion. Repeated treatment with agmatine

2.8. Statistical analysis prevented the deficit in social recognition ability induced by i.n.

MPTP administration, causing a significant reduction in the RID

Data for Neurological Severity Score are shown as median (interquartile

when the familiar juvenile was re-exposed after 30 min (Fig. 3).

range) and comparisons between groups were performed by Kruskal–Wallis non-

parametric test followed by Dunn’s multiple comparison tests. Statistical analysis

of survival curves were performed with the Cox–Mantel test (log-rank) followed 3.3. Effects of agmatine on the general neurological state of aging

by Gehan–Breslow–Wilcoxon test. The rest of data was checked for normality

mice infused intranasally with MPTP

of frequency distribution with the Kolmogorov–Smirnov test and expressed as

mean standard error of mean (S.E.M.). In this case, Student’s t-test and analysis of

± The general neurological state of the animals was evaluated

variance (ANOVA) were applied when appropriate, as informed in the results section

and figure legends. Following significant ANOVAs, multiple post hoc comparisons by the NSS at 13 days after i.n. MPTP administration. As illus-

were performed using the Newman–Keuls test. The accepted level of significance trated in Fig. 4, Kruskal–Wallis non-parametric test followed by

®

for all tests was P 0.05. All tests were performed using the Statistica software

≤ Dunn’s post hoc tests indicated that mice from the control/MPTP

package (Stat Soft Inc., USA).

group scored significantly higher [P 0.05] than the control/control

group. Of high importance, this increase in NSS score induced by

3. Results

i.n. MPTP administration was prevented by the pretreatment with

3.1. Effects of agmatine on the survival rate of aging mice infused

intranasally with MPTP

As can be seen in Fig. 2, log-rank Mantel–Cox test followed

by Gehan–Breslow–Wilcoxon test indicated a lower percentage of

surviving animals in the control/MPTP group when compared to

the control/MPTP group (treatment factor: P 0.05). The statistical

analysis performed point-by-point indicated an increased mortal-

ity (about 50%) of MPTP-infused mice from the 1st to the 13th day

after treatment. Remarkably, repeated treatment with agmatine

(30 mg/kg, i.p.) during 5 consecutive days increased significantly

the 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 of

Fig. 4. Effects of the pretreatment (during 5 consecutive days) with control

aging mice infused intranasally with MPTP

(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 days

The results for the effects of i.n. administration of MPTP after i.n. infusion of control or MPTP (1 mg/nostril). The results are shown as

median (interquartile ranges) of NSS points [control/control (n = 9); control/MPTP

(1 mg/nostril) on the short-term social recognition memory of

(n = 8); agmatine/control (n = 10); and agmatine/MPTP (n = 9)]. *P 0.05 compared

aging mice pretreated with control or agmatine (30 mg/kg) are ≤

to the control/control group. #P 0.05 compared to the control/MPTP group

illustrated in Fig. 3. Two-way ANOVA revealed significant effects for

(Kruskal–Wallis non-parametric test followed by Dunn’s multiple comparison

the pretreatment [F = 10.32, P 0.01], treatment [F = 20.72; tests). 1,33 ≤ 1,33

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 female

C57BL/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 are

expressed 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–Keuls ≤ ≤

test).

agmatine (30 mg/kg, i.p.), suggesting a protective effect of agmatine rearings induced by i.n. MPTP administration was prevented by the

against the neurological impairments induced by MPTP in aging pretreatment with agmatine (Fig. 5D). Therefore, the locomotor

mice (Fig. 4). activity of aging mice was only disrupted at later periods after i.n.

MPTP administration which was prevented by pretreatment with

3.4. Effects of agmatine on the spontaneous locomotor activity of agmatine (30 mg/kg, i.p.).

aging mice infused intranasally with MPTP

The results of locomotor activity of aging mice evaluated for 3.5. Agmatine prevents the loss of dopaminergic neurons induced

5 min in the activity chambers and open field arena at 3 and 19 days, by i.n. MPTP administration in aging mice

respectively, after i.n. MPTP (1 mg/nostril) administration are sum-

marized in Fig. 5. Two-way ANOVA revealed no significant effects With the purpose of determining the relationship between the

for the main factors and their interaction in the total distance trav- motor impairments observed in the open field at later periods after

eled [pretreatment: F1,33 = 1.21; P = 0.28; treatment: F1,33 = 0.45; i.n. MPTP administration in aging mice and dopaminergic cell death

P = 0.48; interaction: F1,33 = 0.28; P = 0.60] (Fig. 5A) and the average in the , the evaluation for TH-positive cells in

speed [pretreatment: F1,33 = 1.31; P = 0.29; treatment: F1,33 = 0.28; the substantia nigra was performed 21 days after i.n. administration

P = 0.60; interaction: F1,33 = 0.32; P = 0.60] (Fig. 5B) evaluated in the of MPTP by immunohistochemistry. Fig. 6A–D shows representa-

activity chambers. tive photomicrographs of TH immunohistochemistry in the ventral

On the other hand, two-way ANOVA revealed significant effects mesencephalon containing SNpc.

for the treatment factor [F = 5.86; P 0.05] and the interaction As can be seen in Fig. 6E, two-way ANOVA revealed signif-

1,29 ≤

between pretreatment and treatment [F = 4.26; P 0.05], but not icant effects for the treatment factor [F1,16 = 8.73; P 0.01] and

1,29 ≤ ≤

for the pretreatment factor [F1,29 = 1.26; P = 0.27], in the number the interaction between pretreatment and treatment [F1,16 = 4.52;

P 0.05], but not for the pretreatment factor [F = 0.52; P = 0.48],

of squares crossed in the open field. Post hoc comparisons indi- ≤ 1,16

cated a significant reduction in the number of crossings in the in the number of TH-positive cells in the SNpc. Subsequent

MPTP/control group that was not observed in the agmatine/MPTP Newman–Keuls tests showed that the i.n. administration of MPTP

group (Fig. 5C). induced a pronounced reduction (about 50%) of TH immunostain-

Regarding the number of rearings, two-way ANOVA revealed ing in the SNpc of aging mice when compared to the control/control

F group (P 0.05). Of high importance, the pretreatment with agma-

significant effects for the main factors [pretreatment: 1,29 = 4.23; ≤

P 0.05; treatment: F = 6.70; P 0.05] and their inter- tine (30 mg/kg, i.p.) was able to attenuate the loss of TH-positive

≤ 1,29 ≤

action [F = 4,23; P 0.05] in this parameter. Subsequent neurons in the SNpc of MPTP-treated mice when compared to con-

1,29 ≤

Newman–Keuls test indicated that the reduction in the number of trol/control group (Fig. 6E).

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

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 substantia

nigra 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 number

2

of TH-positive neurons per mm in SNpc of mice. Values represent the mean SEM (n = 5 animals per group). *P 0.05 compared to the control/control group (two-way

± ≤

ANOVA followed by Newman–Keuls test).

3.6. Agmatine prevents decreased hippocampal glutamate uptake uptake was measured in the hippocampus of aging mice 21 days

induced by i.n. MPTP administration in aging mice after i.n. MPTP administration.

As illustrated in Fig. 7, the i.n. MPTP administration sig-

Glutamate clearance from extracellular space is an important nificantly decrease [F = 5.16; P 0.05] the glutamate uptake

1,14 ≤

mechanism related to the reduction of glutamate excitotoxicity. into the hippocampus of aging mice. The pretreatment with

With the purpose of determining possible alterations in gluta- agmatine did not alter per se the basal hippocampal glutamate

matergic neurotransmission following MPTP treatment, glutamate uptake [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

the CNS including the dopaminergic neurodegeneration induced by

PD-mimetic toxins such as MPTP [30] and rotenone [15,16].

The current data corroborates the neuroprotective potential

of agmatine in PD since it attenuated the dopaminergic cell

loss in the SNpc of aging mice infused intranasally with MPTP

(1 mg/nostril). Moreover, the present study provides the first

preclinical data demonstrating that repeated treatment with agma-

tine (30 mg/kg, i.p.) improves short-term memory and motor

impairments displayed by MPTP-treated aging mice. Finally, the

observed behavioral benefits of exogenous agmatine treatment

were accompanied by the prevention of MPTP-induced decrease

of hippocampal glutamate uptake.

Recent studies performed by our group and others with the

administration of exogenous agmatine in laboratory animals have

Fig. 7. Effects of the pretreatment (during 5 consecutive days) with control (i.p.)

3 identified several relevant functions of this substance that are of

or agmatine (30 mg/kg, i.p.) on hippocampal l-[ H]glutamate uptake in 15-month-

old female C57BL/6 mice infused intranasally with control or MPTP (1 mg/nostril). At potential therapeutic importance, including [17],

the 21th day after i.n. MPTP administration, mice were sacrificed and the hippocam- antinociceptive [28,54,64], anxiolytic [32] and antidepressant-like

pal slices processed for glutamate uptake assay in vitro as described in Section 2.

[73,74] actions. Moreover, age-related changes in agmatine levels

Values are expressed as mean SEM [control/control (n = 4); control/MPTP (n = 3);

± in various brain structures have been demonstrated, thus indicating

agmatine/control (n = 5); and agmatine/MPTP (n = 4)]. P 0.05 compared to the con-

≤ the potential involvement of agmatine in aging process [45].

trol/control group (two-way ANOVA followed by Newman–Keuls test).

In the present study, the repeated treatment with agmatine

(30 mg/kg, i.p.) during 5 consecutive days was able to attenuate

reduction of glutamate uptake [interaction factor: F1,14 = 4.88; significantly the mortality of MPTP-treated aging mice. The acute

P 0.05] (Fig. 7). toxicity effects of MPTP are largely attributed to peripheral mecha-

≤ +

nisms [40]. For instance, MPTP and its toxic metabolite MPP have

been shown to have a variety of peripheral effects including cardiac

3.7. Effects of agmatine on the monoamine oxidase activity

noradrenaline depletion [2], adrenal noradrenaline and dopamine

release [2], hypothermia [27] and neuromuscular blockade via

As can be seen in Fig. 8, Student’s t-tests indicated that the

binding to nicotinic receptors [39]. It must be con-

current tested concentrations of agmatine (0.01–1000 ␮m) had

ceded that, at present stage, it not possible to determine the exact

no significant effect on either MAO-A [t = 1.30, P = 0.25] or MAO-

site of action and molecular mechanisms underlying agmatine

B [t = 0.96, P = 0.38] activities in the mouse brain mitochondrial

homogenates. attenuated the mortality of MPTP-infused aging mice. Neverthe-

less, based on previous literature demonstrating that agmatine

inhibits sympathetically-induced tachycardic responses [14] and

4. Discussion

noradrealine release [60], a speculative hypothesis is that agmatine

may prevent the alterations in the cardiovascular system verified

Therapeutic strategies that slow or stop the neurodegenerative

following MPTP administration.

process of PD are expected to have a major impact on the treatment

Regarding PD symptoms, an increasing number of studies have

of this disease [47]. The current hypothesis about the mechanisms

demonstrated that PD seems to be a multidimensional disease, and

by which neurons come into necrotic or apoptotic process of degen-

besides motor deficits, it is associated with a number of senso-

eration has led to belief that the use of drugs modulating the

rial, cognitive and emotional disturbances that result in a loss in

function of glutamate NMDA receptors may have beneficial effects

quality of of the individuals [13]. In this context, in a recent

in PD therapy [10,63]. In this context, there is increasing evidence

series of studies we demonstrated that a single i.n. infusion of MPTP

of the neuprotective effects of agmatine, which among other possi-

in rodents produces diverse signs of PD such as impairments in

ble targets blockades NMDA receptors, against different insults of

olfactory, 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 of

olfactory bulb to i.n. MPTP toxicity, with a marked reduction in

TH-positive neurons and dopamine depletion occurring 24 h after

i.n. MPTP infusion. On the other hand, these alterations were only

observed later (14–21 days after intransal MPTP administration) in

the SNpc and . Therefore, the existence of compensatory

mechanisms, such as the increase in the number of TH-positive

striatal cells and a downregulation of dopamine uptake in surviv-

ing dopaminergic fibers in the striatum [8], may be responsible for

the lack of striatal changes until 14 days post-MPTP. Consistent

with these observations, the current findings indicated no signifi-

cant alterations in the total distance traveled and the average speed

by aging mice in the activity chambers at 3 days after i.n. MPTP

administration.

Since the i.n. administration of MPTP does not cause, at least at

initial periods, gross motor alterations that would preclude assess-

Fig. 8. Effects of agmatine on mouse brain mitochondrial MAO-A and MAO-B activ-

ment of cognitive functions, we investigated the impact of i.n.

ities in vitro. Agmatine was tested in a concentration range of 0.01–1000 ␮m. The

MPTP administration on social recognition memory of aging mice.

values represent the mean S.E.M. of three individuals experiments, performed in

±

duplicate. Aging mice infused with MPTP (1 mg/nostril) spent significantly

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

more time investigating the juvenile during the second presenta- preventing the locomotor impairments in the open field and the

tion than they did in the first encounter, suggesting an impaired decrease of the TH immunoreactivity in the SNpc induced by i.n.

ability to recognize the juvenile mouse after a short time. Previ- infusion of MPTP in aging mice. These results corroborate recent

ous studies from our group have demonstrated that rodents treated findings on agmatine neuroprotection in cellular models of PD-like

intranasally with MPTP performed normally in the long-term reten- neurodegeneration [15,16]. Moreover, Gilad et al. [30] published

tion session (24 h after training) of the inhibitory avoidance task a pioneer study demonstrating that the treatment with agmatine

[12,58] and in the spatial reference memory version of the water (100 mg/kg, i.p.) during 5 days attenuated MPTP-induced reduction

maze [59]. In contrast, MPTP-infused animals displayed a poor per- of synaptosomal dopamine uptake when administered 8 h after

formance in the short-term retention session (1.5 h after training) MPTP injection (40 mg/kg, i.p., for 2 days). Taken together, these

of the inhibitory avoidance task [12,58] as well as in the working results suggest that agmatine may represent a potential disease-

memory version of the water maze [59]. These findings are consis- modifying therapy for PD.

tent with the view of human studies suggesting that PD patients It is well known that MAO-B inhibition reduces the generation

+

present cognitive deficits mainly in working memory and short- of the MPP from MPTP, protecting against the dopaminergic cell

term memory tasks with long-term spatial (declarative) death in the SNpc [36]. In accordance with previous literature [30],

mostly spared [11]. we observed that agmatine does not interfere with MAO-B activity

Of high importance, the present findings demonstrate that the on mouse brain mitochondrial homogenates, indicating that MAO-

+

pretreatment with agmatine (30 mg/kg, i.p.) during 5 consecutive B-catalyzed conversion of MPTP to MPP is not affected. However,

+

days was able to prevent the short-term social memory deficits the evaluation of the time-course of MPP kinetic in the mouse

of MPTP-treated aging mice. Therefore, from these limited results brain after i.n. MPTP administration constitutes a very interesting

it appears that agmatine might be particularly useful to restore field that requires additional research.

memory processes in PD. The current results are in accord with The neuroprotective effects of agmatine may result from dif-

previous findings indicating the cognitive-enhancing properties of ferent mechanisms including blocking of NMDA receptors [70],

agmatine in diverse animal models of brain injury. For instance, the inhibition of synthase (NOS) [29],

repeated administration of agmatine (5–40 mg/kg, i.p.) prevented scavenging [6] and protection against mitochondrial membrane

and memory impairments in rodents induced by infusion potential collapse [4–6]. However, the sequence of events lead-

of aggregated beta-amyloid(25-35) [7], lipopolysaccha- ing to the protective effects of agmatine against cell damage has

ride [71], streptozotocin [9] and [49]. Moreover, not been fully elucidated. Here we observed that agmatine was

recent studies [44,45,67] have demonstrated that spatial learning able to prevent the decrease of hippocampal glutamate uptake in

in rodents induces elevation in agmatine levels at in the aging mice following i.n. MPTP administration. Corroborating our

rat hippocampus, providing further evidence of its participation in findings, previous studies [18,35] have demonstrated that MPTP

learning and memory processes. decreases glutamate uptake by astrocytes in cell culture. Therefore,

Additionally, in the present study we observed a later (19 days one possible mechanism by which agmatine may exert protective

after i.n. MPTP administration) reduction in the locomotor activ- effects against MPTP neurotoxicity may be due to the modulation of

ity of MPTP-treated 15-month-old female C57BL/6 mice evaluated glutamate into neural cells, the main mechanism respon-

in the open field that was accompanied by a marked reduction sible for decreasing extracellular glutamate levels, thus attenuating

(about 50%) of TH-positive neurons in the SNpc. These findings glutamate neurotoxicity.

contrast with our previous study demonstrating that young adult

(6-months-old) male C57BL/6 mice infused intranasally with the

same dose of MPTP do not present gross motor alterations [58]. 5. Conclusions

Therefore, the current data indicates that the age and gender of

the animals represent important factors that modulate the appear- The present findings reinforce the i.n. MPTP administration as a

ance of motor symptoms in the i.n. MPTP model of PD. Reinforcing valuable rodent model for testing novel palliative and neuroprotec-

the current findings, previous studies have demonstrated that tive compounds for PD and demonstrate that the age of the animals

dopamine-depleting effects and motor impairments induced by represent an important factor that modulate the appearance of

i.p. MPTP administration in mice are age- [41,52] and gender- motor symptoms in this model. More importantly, the present

dependent [3,26,41,53,68]. study provides the first preclinical data indicating that repeated

Epidemiological studies have shown a prevalence of PD in men systemic treatment with agmatine prevents short-term memory

compared to women [20]. In women, the age at onset of PD cor- and motor impairments as well as dopaminergic cell loss in the

relates with the end of the fertile life [62]. However, results from SNpc of aging mice submitted to an experimental model of PD.

previous preclinical studies addressing gender-related differences These results provide new insights in experimental models of PD,

on MPTP toxicity have been inconsistent, with increased suscep- indicating that agmatine may represent a new therapeutic tool for

tibility to MPTP-induced behavioral and neurochemical changes the management of cognitive and motor symptoms of PD, together

been described for both male [3,26] and female [41,53,68] mice. with its neuroprotective potential.

Interestingly, Unzeta et al. [68] observed significant differences in

MAO-A and MAO-B activities during the oestrous cycle as well as

between adult male and female mice. Since the neurotoxic effects Acknowledgements

+

of MPTP depend on its conversion to the MPP by MAO-B, the

observed differences in MAO-B activity may be involved in the This work was supported by grants from Conselho Nacional

gender-related effects of MPTP. Therefore, the use of female mice de Desenvolvimento Científico e Tecnológico (CNPq), Coordenac¸ ão

with 15-months-old in the present study, which at this age show de Aperfeic¸ oamento de Pessoal de Nível Superior (CAPES), Pro-

cessation of estrous cycle with very low levels of estrogen [24], grama de Apoio aos Núcleos de Excelência (PRONEX – Project

attenuates the hormonal variability which could interfere with NENASC), Fundac¸ ão de Apoio à Pesquisa do Estado de Santa Cata-

MPTP toxicity. rina (FAPESC), FINEP (Financiadora de Estudos e Projetos-IBN-Net

Of high importance, the administration of agmatine demon- #01.06.0842-00) and INCT (Instituto Nacional de Ciência e Tec-

strated once again its neuroprotective properties as previously nologia) for Excitotoxicity and Neuroprotection. FCM and AAC

described in several models of neuronal damage [31,42,72], receive 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 no [25] Flierl MA, Stahel PF, Beauchamp KM, Morgan SJ, Smith WR, Shohami E. Mouse

closed head injury model induced by a weight-drop device. Protocols

financial or personal conflicts of interest related to this work.

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