1

Chronic atrazine exposure causes disruption of the spontaneous 1

locomotor activity and alters the striatal dopaminergic system of 2

the male Sprague-Dawley rat 3

4

Ulises Bardullas, Magda Giordano, Verónica M. Rodríguez * 5

Departamento de Neurobiología Conductual y Cognitiva, 6

Instituto de Neurobiología, Universidad Nacional Autónoma de México, 7

Boulevard Juriquilla 3001, Querétaro, Querétaro, 76230, México. 8

Phone: 52-442-2381061 9

Fax: 52-442-2381046 10

* To whom correspondence should be addressed at Departamento de 11

Neurobiología Conductual y Cognitiva, Instituto de Neurobiología, Universidad 12

Nacional Autónoma de México, Boulevard Juriquilla 3001, Querétaro, Querétaro, 13

76230, México. Phone: 52-442-2381061 Fax: 52-442-2381046 14

E-mail address: vermire@yahoo.com; vmrodriguez@comunidad.unam.mx 15

16

Short Title: Atrazine and dopaminergic systems17

2

ABSTRACT 1

The herbicide atrazine (ATR) is widely used around the world, and is a 2

potential toxicant of the dopaminergic systems. Nigrostriatal and mesolimbic 3

systems are the two major dopaminergic pathways of the central nervous system; 4

they play key roles mediating a wide array of critical motor and cognitive functions. 5

We evaluated the effects of exposing male rats for one year to 10 mg ATR/kg B.W. 6

on these systems using motor and cognitive tasks and measuring monoamine 7

content in the striatum, nucleus accumbens, prefrontal cortex, and hypothalamus. 8

ATR administration resulted in impaired motor coordination and greater 9

spontaneous locomotor activity only after 10 to 12 months of exposure. Chronic 10

exposure to 10 mg ATR decreased striatal dopamine, but had no effect on 11

accumbal, hypothalamic or cortical monoamine content. Chronic ATR exposure 12

caused discrete changes in learning tasks that involve either the striatum or the 13

nucleus accumbens. These results indicate that chronic exposure to ATR 14

preferentially targets the nigrostriatal dopaminergic pathway, in comparison to the 15

other dopaminergic pathways evaluated in this study, inducing behavioral and 16

neurochemical alterations. In order to unveil the full extent of atrazine’s effects on 17

the nervous system, other neurochemical systems should be considered in future 18

studies. 19

20

Keywords: Dopamine; herbicides; striatum; neurotoxicity; behavior; endocrine 21

disruptors. 22

23

24

3

1. Introduction 1

The herbicide ATR (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) is 2

extensively used around the world, mainly to control annual grasses and broadleaf 3

weeds in crops like corn, sorghum and sugarcane. The mode of action of ATR in 4

plants involves the inhibition of photosynthesis by preventing electron transfer at 5

the reducing site of complex II in the chloroplast [26] ATR is also the most 6

commonly found herbicide in surface waters and deep-wells in areas of intense 7

usage [1]. Recent reports indicate that the risk of ATR exposure is not only 8

occupational, since it is also found in food and houses of farming and non-farming 9

families in rural areas [11,23,35]. 10

ATR is a well-known endocrine disruptor, inducing hermaphroditism in male 11

frogs exposed to doses as low as 0.1 mg ATR/L of water [27]. Studies using Wistar 12

rats exposed to a range of doses from 12.5 to 300 mg ATR/kg of body weight 13

(B.W.) report changes in ACTH concentrations [33]; inhibition of testosterone 14

production was reported in juvenile male rats treated with 50 mg ATR/kg B.W. per 15

day by gavage (from postnatal day 46 to 48) or chronically (from postnatal day 22 16

to 48) [22]. Reproductive system alterations were observed in male Long-Evans 17

rats after gestational exposure to ATR (gestational days 15-19, dams received 100 18

mg ATR/kg B.W.) [44]. While dose-dependent decreases of prolactin (PRL) and 19

luteinizing hormone (LH) release in female Long-Evans rats exposed via gavage to 20

50-300 mg ATR/kg/day for 1, 3, or 21 days were found [10,53]; these reductions in 21

PRL and LH could be related to increases in dopamine (DA) content in the median 22

eminence and preoptic areas of the hypothalamus [9]. 23

4

In spite of the environmental relevance of ATR exposure, few studies have 1

evaluated the neurotoxicity of ATR. It has been reported that ATR crosses the 2

blood brain barrier and enters the brain through an unknown mechanism [49]. 3

Similar levels of 14C-atrazine have been found in the anterior and posterior 4

hypothalamus and striatum of nursing rats treated with a single dose of 2 or 4 5

mg/kg by gavage on postnatal day 3 [52]. ATR has been identified as a potential 6

dopaminergic system toxicant in both in vivo [7,18,48] and in vitro studies [12-14]. 7

Chronic exposure (six months) to 10 mg ATR/kg B.W. or subchronic exposure (14 8

days) to 125 or 250 mg ATR/kg B.W. results in a lower striatal DA content and 9

produces a loss of dopaminergic neurons in both the substantia nigra pars 10

compacta (SNpc) and the ventral tegmental area (VTA) in male Long-Evans rats 11

[48] and C57BL/6 juvenile male mice [7]. Incubation of striatal tissue slices from 12

adult male Sprague-Dawley rats with up to 500 µM of ATR for 4 hours produces a 13

dose-dependent decrease in total DA and increase in DA turnover, while in the 14

conditioned media a dose-dependent increase in DA levels with a consequent 15

dose-dependent decrease in DA turnover was observed [18]. It has also been 16

reported that incubation with 1-250 µM ATR causes a dose-dependent reduction in 17

DA uptake into striatal vesicles from Sprague-Dawley rats, due to a significant 18

decrease in the Vmax, without alterations of the Km value or of the vesicular 19

transporter, that could lead to elevated cytosolic DA in the presynaptic terminal 20

[28]. 21

In addition to the dopaminergic changes related to ATR exposure, it has 22

been reported that exposure to low, ecological ATR doses of 1 to 100 µg/kg per 23

day from gestational day 14 to postnatal day 21 produces a dimorphic 24

5

neurodegeneration pattern with damage to axonal processes and perikarya in the 1

female but not male hippocampal and hypothalamic regions, and reduction in the 2

total number of neurons. Also, hypothalamic upregulation and a trend towards 3

downregulation of striatal levels of somatostatin receptor subtype 2 mRNA 4

expressing neurons was observed in female mice. Meanwhile, in male mice 5

upregulation of neurons expressing somatostatin receptor subtype 3 mRNA in 6

hypothalamus and amygdala, and downregulation of this receptor in cortical and 7

hippocampal areas was detected [24]. ATR seems to modulate the GABAergic 8

system as well; it inhibits the binding of Ro15-4513 (an agonist of the 9

benzodiazepine binding site in the GABAA receptor) to cortical membranes 10

obtained from Long-Evans rats when incubated with 1 – 400 M of ATR [51]. By 11

targeting all these neurotransmitter systems, ATR exposure has the potential to 12

disrupt the planning and regulation of voluntary movements [3] and diverse aspects 13

of memory [19,20]. 14

At present, there are only two reports in the literature that assess the effects 15

of ATR exposure on locomotor activity in male rats. One of them found less vertical 16

exploration after exposure to a high dose (1 g ATR/kg B.W.) for 4 -11 days [56]. 17

The other study found that 10 mg ATR/kg B.W. administered daily for six months, 18

produced hyperactivity on the horizontal plane, while the low dose (5 mg ATR/kg 19

B.W.) did not show any alterations on horizontal locomotor activity [48]. Assessing 20

the effects of ATR exposure on locomotor activity has been shown to be useful for 21

evaluating the integrity of the nigrostriatal pathway, but it is unknown if ATR can 22

alter other functional aspects of both the nigrostriatal and mesolimbic pathways. If 23

6

ATR exposure disrupts several neurotransmitter systems as stated above, it can 1

impair brain functions associated with those systems. Monoamines, especially 2

dopamine, are known to modulate motor as well as cognitive functions in the 3

central nervous system [21,25,50]. Thus, in order to evaluate if ATR exposure can 4

alter brain function, animals were tested on a battery of behavioral tests that 5

included, motor, sensorimotor, and learning tasks. The specific aims of this study 6

were: (i) to determine the onset and the nature of the alterations in locomotor 7

activity after chronic ATR exposure, (ii) to evaluate possible effects of ATR on 8

learning tasks, (iii) to establish whether these behavioral alterations correlate with 9

changes in the monoamine content in brain regions that receive afferents from the 10

nigrostriatal and mesolimbic pathways, and (iv) to evaluate the effects of ATR on 11

hypothalamic monoamine levels given the results of previous studies suggesting 12

this area as a target. 13

14

15

16

7

2. Materials and methods 1

2.1. Subjects and chemicals. 2

Twenty, 21-day-old male Sprague-Dawley rats were obtained from the 3

animal care facility of the Instituto de Neurobiologia-UNAM and kept under a 12-h 4

inverted dark/light cycle (lights on at 20:00) at constant temperature (23 ± 5°C). 5

Experiments were carried out according to the Norma Oficial Mexicana de la 6

Secretaría de Agricultura (SAGARPA NOM-062-ZOO-1999), which complies with 7

the guidelines in the Institutional Animal Care and Use Committee Guidebook (NIH 8

Publication 80-23, Bethesda, MD, USA, 1996) and were approved by the local 9

Committee on Bioethics. 10

Atrazine was purchased from Chem Service (West Chester, PA, USA). 11

Reagents for high performance liquid chromatography (HPLC) were purchased 12

from Sigma-Aldrich (St. Louis, MO, USA). 13

14

2.2. Treatment 15

Ten animals received a daily dose of 10 mg ATR/kg B.W. in hand-made 16

pellets, while ten control rats received pellets without ATR. These pellets were 17

supplemented with control diet provided ad libitum until the animals reached 300 g; 18

body weight was recorded weekly. Once animals reached 300 g, both control and 19

ATR groups were maintained at this body weight by caloric restriction for the 20

duration of the experiment. Behavioral assessments took place monthly or 21

bimonthly during the twelve months of ATR exposure (see below). The 22

8

experimental design and the timeline for the present study are represented in Fig. 1

1. 2

Locomotor activity was evaluated throughout the period of ATR exposure; 3

after the last locomotor activity session, rats were euthanized by decapitation, the 4

brain was extracted, and left and right striatum, nucleus accumbens (NAC), frontal 5

cortices, and hypothalamus were dissected on ice and frozen at −80 °C to 6

measure DA, serotonin (5-HT) and their metabolites, and to evaluate the levels of 7

tyrosine hydroxylase (TH) in striatum and nucleus accumbens. 8

9

2.2.1 Food preparation. One kilogram of chow diet (Purina, PMI international 10

Richmond, Indiana, USA) previously moisturized with deionized water was 11

thoroughly mixed with 300 mg ATR (Chem Service). From this dough, pellets were 12

confectioned and deposited in an oven set at 50 °C until they were perfectly dry 13

(recovery 97-103%). 14

15

2.3. Spontaneous locomotor activity. 16

Each rat was individually placed in an automated locomotor activity chamber 17

equipped with horizontal and vertical infrared beams (Accuscan Instruments Inc., 18

Columbus, OH, USA). Locomotor activity was recorded, and data were collected 19

over the course of a 25-h session (12 h light: 12 h dark). The first hour, which is 20

usually when the greatest activity is found, was analyzed separately from the 21

remaining 24 hours. Food and water were available ad libitum during this session. 22

Data was collected monthly for months 1 - 6 and bimonthly thereafter. The activity 23

chambers record a variety of movement parameters that can be classified as: 24

9

horizontal variables (horizontal activity, total distance, number of movements and 1

movement time); vertical variables (vertical activity, and vertical time), stereotyped 2

behavior (stereotypy counts, number of stereotypy, and stereotypy time); and 3

location parameters (margin distance and center distance). For this task 7-9 rats 4

per group were tested for the duration of the experiment. 5

6

7

2.4. Motor coordination assessment. 8

At 10 months of ATR exposure rats were pretrained on an automated 9

rotorod unit (IITC Life Science, Woodland Hills, CA, USA), with five, 3.75-inch-10

diameter drums, which were modified by adding a rough surface to improve the 11

rat’s grip, using the acceleration protocol previously described by Monville et al. 12

[38]. Briefly, rats were trained twice a day during three days with a smooth 13

acceleration rate (from 4 to 20 rpm in 60 sec) until they were able to stay on the 14

rod. Any animal that did not fulfill this requirement was discarded from this test; two 15

rats of each treatment were thus discarded. After the training phase, the animal 16

received three consecutive daily trials. The rotorod was programmed to accelerate 17

from 10 to 45 rpm in 180 seconds, and the total time of evaluation was 240 18

seconds. Latency and acceleration to fall were registered automatically by a trip 19

switch under the floor of each rotating drum. For this task 7 rats per group were 20

tested. 21

22

2.5. Learning tasks 23

10

Spontaneous alternation. A plus maze was used to evaluate spontaneous and 1

delayed alternation. It was built of black acrylic, and consisted of a central area (12 2

X 12 cm) surrounded by four arms (length 48 X height 32 X wide 12 cm), each with 3

a cylindrical cup (2.4 cm in diameter) at its end, where the reinforcer (a 100 mg 4

sugar pellet) was found. One arm was always closed in order to form a t-maze. 5

Animals were allowed to explore the maze for five min on one day without access 6

to the reinforcer. After the habituation period, rats were tested eight times a day for 7

four days. The maze was cleaned between trials with a weak solution of acetic acid 8

(1%). Latency to reach the reinforcer, and the arm selected were recorded. This 9

learning task took place at 11 months of ATR exposure. For this task 8 rats per 10

group were used. 11

Delayed alternation. After the spontaneous alternation test, rats were trained on a 12

delayed alternation task, as described by others [47]. Briefly, during the first trial of 13

each day, sugar pellets were present in both arms. On the following trials, the arm 14

opposite to the one the rat had chosen in the previous trial was baited (the other 15

arm was unbaited), except when the animal had gone into the empty arm on the 16

last trial. There was a delay of 1 min between each trial. When the rat entered an 17

unbaited arm, an error was recorded. Seven trials per session were given over the 18

course of 5 days. This test was carried out at 11 months of ATR exposure. For this 19

task 7 - 8 rats per group were used. 20

Eight-arm radial maze. For the win-shift evaluation, we used a radial maze made 21

of acrylic with an octagonal central area (28-cm diameter) connected to eight arms 22

(58 X 10.5 X 10.5 cm), with a cylindrical cup (2.4 cm in diameter) at the end of 23

each arm. The maze was elevated 80 cm from the floor, and there were many 24

11

spatial cues in the testing room (doors, the experimenter, posters, etc.) under soft 1

illumination (40 W). 2

Win-shift: Rats were allowed to explore the maze (habituation session) for five 3

minutes on one day. After this habituation period, rats were trained on a win-shift 4

task as described previously [47]. Briefly, rats were allowed up to five minutes to 5

retrieve all sugar pellets from the cups at the end of each arm. When a rat re-6

entered a previously visited arm, an error was recorded. The maze was cleaned 7

between trials with a weak solution of acetic acid (1%). One trial was given on each 8

of three consecutive days. This test took place at 6 and 10 months of ATR 9

exposure. For this task 9 rats per group were used. 10

Non-delayed random foraging paradigm. This task was modified from Floresco 11

et al. [19]. Briefly, rats were required to forage for sugar pellets placed at random in 12

the food cups of 4 of the 8 arms. A novel set of arms was baited each day. Each 13

rat was allowed up to five minutes to retrieve the pellets and return to its home 14

cage. The maze was cleaned between trials with a weak solution of acetic acid 15

(1%). When a rat re-entered a previously visited arm an error was recorded. This 16

test was carried out at 12 months of ATR exposure. One trial was given on each of 17

five consecutive days. For this task 6 rats per group were used. 18

19

2.6. Determination of monoamines and their metabolites. 20

Striatum, nucleus accumbens, prefrontal cortex, and hypothalamus were 21

collected separately and disrupted by sonication in 0.1 M perchloric acid. 22

Homogenates were centrifuged at 10,000 g for 40 min, and supernatants were 23

frozen at −80 °C until quantification. Pellets were digested in 0.5 M NaOH, and 24

12

protein levels were quantified using the Bradford technique. DA, its metabolite 1

dihydroxyphenylacetic acid (DOPAC), 5-HT and its metabolite 5-hydroxyindole 2

acetic acid were measured by HPLC with electrochemical detection as described 3

elsewhere [2]. Briefly, a PerkinElmer pump series 200 (Waltham, MA, USA) was 4

joined to a chromatographic column (Grace Davison Discovery Sciences, 5

Deerfield, IL, USA) packed with a catecholamine adsorbosphere (3-μm particle 6

size, 100×4.8 mm). An electrochemical detector Bioanalytical system, LC-4C (West 7

Lafayette, IN, USA) was coupled to the system. The amperometric potential was 8

set at 850 mV relative to the silver/ silver chloride electrode, and the sensitivity of 9

the detector was set at 2 ηA. The mobile phase was an aqueous, isocratic, 0.1 M 10

monobasic phosphate solution containing 0.5 mM sodium octyl sulfate, 0.03 mM 11

EDTA, and 12 – 14% (vol/vol) methanol. The results were analyzed with the 12

TotalChrom Navigator version 6.3.1.0504 (PerkinElmer) and are expressed in 13

ng/mg tissue protein. DA turnover was expressed as the ratio of DOPAC to DA, an 14

index of DA utilization. 15

16

2.7. SDS-PAGE and Western blot of TH. 17

The striatal and nucleus accumbens tissues were placed in buffer 18

containing 5mM Tris-HCl, 1mM EDTA and 1% SDS. Samples were sonicated for 3 19

seconds on ice. Protein concentration was determined using a Bio-Rad DC protein 20

assay (Hercules, CA, USA). Three micrograms from each sample were loaded and 21

running in 5 – 12% SDS-PAGE. Proteins were transferred to PVDF membranes for 22

1 h with semi-dry blotting system (Bio-Rad). Membranes were blocked overnight at 23

4º C (1% TBS-tween-5% Blotto). Protein of interest was detected using an anti-TH 24

13

antibody (Chemicon International Inc., Temecula, CA, USA), followed by anti--1

tubulin (DSHB University of Iowa, Iowa City, IA, USA) antibody as a control for 2

protein load. After secondary antibody incubation, membranes were visualized 3

through chemiluminescense (ECL, Amersham, USA) and documented with a 4

Molecular Dynamics STORM 860 scanner (Sunnyvale, CA, USA). 5

6

7

2.8. Statistical analysis. 8

Spontaneous locomotor activity, body weight, motor coordination 9

assessment, and learning tasks were analyzed using two-way repeated-measures 10

analysis of variance followed by post-hoc tests (Student’s t-test) in the case of 11

significant main effects or interactions. Non-paired t-tests, with Bonferroni 12

correction were used to compare overall mean activity during the dark or the light 13

phases of the dark-light cycle. Multivariate analysis of variance (MANOVA) 14

followed by univariate tests was calculated for monoamine levels per brain region. 15

Multiple linear regression analysis was used to evaluate if monoamine levels per 16

region significantly predicted activity during the last month of testing. Data 17

analyses were carried out using StatView version 5.0 (SAS Institute Inc., Cary NC 18

USA) and SPSS version 17.0.0 (SPSS Inc., Chicago IL USA) software. Statistical 19

significance was defined as p < 0.05. 20

21

14

3. Results 1

3.1. Body weight and general appearance 2

Rats exposed to 10 mg ATR/kg B.W. for one year did not differ in their 3

general appearance or body weight from the control group on the scheduled 4

evaluations (data not shown), as reported previously [48]. One animal per group 5

died during the first six months of ATR exposure. 6

7

3.2. Spontaneous locomotor activity 8

From the first to the sixth month of ATR exposure, no differences between 9

groups were observed on spontaneous locomotor activity. However, in all monthly 10

recordings there was a significant effect of sampling time during the 24-h recording 11

period, with the characteristic rodent pattern of hyperactivity during the dark phase 12

of the dark-light cycle (data not shown). 13

During the initial 1-hour, no significant effects of ATR treatment were found 14

on horizontal activity, vertical activity, or stereotypy counts, but there were 15

significant effects of duration of ATR exposure [F´s (4, 56) = 3.42 – 12.18, p = 0.01 16

- 0.0001], and of interaction (treatment x duration of ATR exposure) [F´s (4, 56) = 17

2.51 – 6.13, p = 0.038 - 0.005]. The ATR group was significantly less active in the 18

horizontal and vertical planes, and stereotypy counts were lower on the eighth 19

month of ATR exposure [t´s (14) = - 2.702 – - 3.534, p = 0.019 - 0.003]; in contrast, 20

after one year of ATR exposure, the horizontal activity and stereotypy counts 21

increased significantly [t´s (14) = 4.861 – 4.909, p = 0.0004 - 0.0003], as shown in 22

Fig. 2. 23

15

Spontaneous 24-h locomotor activity was altered by ATR exposure. Fig. 3 1

shows the mean activity of the ATR group in all measures of locomotor activity as 2

percentage difference from the control group during the dark, and light phases of 3

the light-dark cycle, at 8, 10 and 12 months of ATR exposure. At eight months of 4

ATR treatment the number and time of stereotypies were lower during the light 5

phase of the daily cycle [t´s (16) = - 4.196 – - 4.092, p = 0.0009 - 0.0007] (Fig. 3A’). 6

No significant alterations of spontaneous locomotor activity parameters were found 7

at 10 months of ATR treatment (Figs. 3B, B’). And after 12 months of ATR 8

exposure significant hyperactivity was found during both the dark [t´s (15) = 3.934 9

– 5.845, p = 0.002 - 0.0001] and light phases [t´s (15) = 3.870 – 5.266, p = 0.001 - 10

0.0002] in several parameters of locomotor activity (see Figs 3C, C’). 11

Regarding the pattern of spontaneous 24-h locomotor activity at 12 months 12

of ATR exposure, Fig. 4 shows two representative measures, total distance and 13

horizontal activity, over a 24-hour period in blocks of three hours. There were 14

significant effects of ATR treatment [F´s (1, 15) = 21.584 – 25.513, p = 0.0002 - 15

0.0003], of sampling time [F´s (7, 105) = 12.226 – 13.826, p = 0.0001], and also 16

significant interaction effects (treatment X sampling time) [F´s (7, 105) = 3.676 – 17

4.752, p = 0.0001 - 0.001]. The results of the post-hoc analyses are shown in Fig. 18

4A and 4B. 19

20

3.3. Motor coordination test 21

Significant effects of ATR treatment were found on the tenth month of ATR 22

exposure in motor coordination using the rotorod acceleration protocol [F (1, 12) = 23

4.848, p = 0.048]; there were no effects of session or interaction. The control group 24

16

showed a consistent increase in latency to fall off the rod on days 2 and 3 of 1

testing, whereas rats exposed to 10 mg ATR/kg showed a relatively stable 2

performance. The difference between groups was evident on sessions 2 and 3 [t´s 3

(12) = - 2.249, - 2.334, respectively, p = 0.044-0.037] (Fig. 5). 4

5

3.4. Learning and memory tasks 6

Spontaneous alternation: There was a significant effect of ATR treatment [F (1, 14) 7

= 10.63, p = 0.0057] and of interaction (ATR treatment X session) [F (3, 42) = 8

3.548, p = 0.022] on spontaneous alternation. Post-hoc analysis showed that the 9

ATR group made more errors in session 3 [(t = (14) = 4.49, p = 0.0005]. (See 10

supplementary Fig. 1). 11

Delayed alternation: No significant effects of ATR treatment or interaction 12

(treatment X session) were found, but there was a significant effect of days of 13

training [F (4, 52) = 8.21, p < 0.0001], showing that both groups learned the task. 14

(See supplementary Fig. 2). 15

Win-shift task: No significant group effects were found on the number of errors 16

made or on the time to solve either task on an eight-arm radial maze. There was 17

only a significant effect of days of training on the number of errors in the win-shift 18

task [F (2, 28) = 3.38, p = 0.0463]. (See supplementary Fig. 3). 19

Non-delayed random foraging paradigm: There was a significant treatment effect 20

on number of errors made [F (1, 10) = 7.097, p = 0.0237], and significant effects of 21

days of training on latencies [F (4, 40) = 33.850, p < 0.0001] (Fig. 6). A detailed 22

analysis of the type of errors made showed significant treatment effects on baited-23

arm reentries [F (1, 10) = 7.097, p = 0.0300] but not on non-baited arm reentries. 24

Comentario [MG1]: No coincide con lo que se dice en los métodos, ver comentario.

17

Post-hoc analyses showed an increase in the total number of errors in the ATR 1

group in session 4 [t (10) = -2.267, p = 0.0468]; also the number of errors for baited 2

arm reentries was significantly greater [t (10) = - 2.467, p = 0.0344] in the same 3

session, and a trend for an increase on the number of errors for baited arm 4

reentries was found in session 5 [t (10) = - 2.071, p = 0.0652] (Fig. 6). 5

6

3.5. Effects of chronic ATR exposure on brain content of monoamines and their 7

metabolites. 8

The Hotelling’s T-square multivariate test of overall differences among 9

groups was not statistically significant for any of the regions evaluated. The 10

univariate between-subjects tests showed that dopamine levels were significantly 11

related to ATR exposure in the striatum [F (1, 16) = 5.11, p = 0.038; eta squared = 12

0.24]. Although dopamine levels in the hypothalamus increased, this change was 13

not statistically significant (p = 0.08). These results indicate that not all 14

monoamines and metabolites measured were altered by ATR exposure, on the 15

contrary, the effect was selective for dopamine in striatum (see Table 1). 16

The multiple regression analysis calculated for monoamine content per brain 17

region, was significant for no other region but the striatum [F (1, 16) = 5.07, p = 18

0.039]. From all monoamines, only dopamine was a significant predictor of 19

locomotor activity (horizontal activity) during the last month of exposure [B =-12.68; 20

t (16) = - 2.253; p = 0.039] (Fig. 7). 21

22

3.6. Total TH protein levels. 23

18

No significant effects of ATR treatment were found for the content of the 1

protein tyrosine hydroxylase in striatum [t (16) = - .297, p > 0.05] or nucleus 2

accumbens [t (11) = - 0.362, p > 0.05]. 3

4

3. Discussion 5

Exposure to pesticides such as rotenone (systemic infusion of 2-3 mg/kg 6

during 7-36 days to male Lewis rats), and the combination of paraquat and maneb 7

(paraquat 10 mg/kg plus maneb 30 mg/kg twice a week for six weeks) [4,55] have 8

been demonstrated to alter the nigrostriatal dopaminergic system in murine models 9

reproducing the phenotypic characteristics of Parkinson´s disease. Recently, 10

studies with rats exposed to 10 mg ATR/kg B.W. during six months and C57 male 11

mice exposed to 5, 25, 125, or 250 mg ATR/kg B.W. for 14 days [7,48], have 12

pinpointed ATR as a potential basal ganglia toxicant altering dopaminergic 13

physiology. However, little is known about the behavioral aspects associated with 14

ATR exposure, and its effects on the dopaminergic system. 15

Rodriguez et al. [48] found that male Long-Evans rats treated with 10 mg 16

ATR/kg B.W. during six months, were hyperactive and showed decrements on 17

striatal dopamine when exposed to 10 mg ATR/kg B.W. They found no effects 18

when animals were treated with 5 mg ATR/kg B.W. In agreement with those 19

earlier results, the present study showed that chronic exposure to 10 mg ATR/kg 20

B.W. for one year induced multiple changes in motor activity, decrements in striatal 21

dopamine and minor alterations of cognitive functions involving the nigrostriatal 22

and mesolimbic pathways. Since only one dose of ATR was used, there is not 23

19

enough information to ascertain if this is the minimal dose at which ATR exposure 1

could alter dopamine content, or behavior; nevertheless our results suggest that 2

chronic exposure to 10 mg ATR/kg B.W. is sufficient to induce neurochemical and 3

behavioral changes in Sprague-Dawley rats. 4

The evaluation of the initial hour of locomotor activity showed biphasic 5

alterations of the horizontal, vertical, and stereotypic parameters of locomotor 6

activity in the ATR group. It appears that exposure to this herbicide disrupts the 7

characteristic pattern of diminishing exploration of a known environment over the 8

course of monthly evaluations. Such alterations are consistent with the 24-hour 9

recordings, where a similar biphasic effect was observed. It is important to note 10

that decreases or increases in locomotor activity found at 8 or 12 months of ATR 11

exposure, respectively could be missed when only one observation is scheduled, 12

emphasizing the need for a closer and longer evaluation of this behavior. This 13

particular pattern of behavioral changes suggests that the effects of ATR on 14

underlying neural circuits differ according to duration of exposure. After an initial 15

period of ATR exposure, hypoactivity is observed, and continued exposure results 16

in robust hyperactivity, and perseverative behavior. These observations indicate 17

that ATR affects neuronal function, and as a consequence it disrupts neural circuits 18

underlying spontaneous motor activity, and learning. Our analysis of the 19

monoaminergic systems, indicates that circuits regulated by dopamine may be the 20

ones targeted by ATR exposure. Indeed dopaminergic circuits are known to 21

participate in learning and motor tasks [21,25,50]. The pattern of behavioral 22

changes may be the result of adjustments in neural function, due to the metabolic 23

and neurochemical challenges imposed by ATR. 24

20

In plants the mode of action of ATR involves the inhibition of photosynthesis 1

by preventing electron transfer [26], but not much is known about the mode of 2

action of ATR in mammalian cells. A recent study by Lim et al. [34] showed that 3

ATR interferes with electron transfer through the oxidative phosphorylation 4

complex at Q sites in mitochondria, resulting in reduced oxygen consumption in 5

soleus muscle or liver mitochondria of male Sprague-Dawley rats exposed during 5 6

months to 30 or 300 mg ATR/kg B.W. Filipov et al. [18] found that ATR 500 μM 7

decreased tissue DA levels in striatal slices incubated for 4 h. Based on their 8

pharmacological manipulations they suggest that ATR may be interfering with the 9

vesicular storage or cellular uptake of DA. TH activity was not affected. In a later 10

study, the same group [28] investigated the effects of in vitro ATR (1-250 microM) 11

exposure on DA uptake using isolated rat striatal synaptosomes and synaptic 12

vesicles. They observed that ATR inhibited DA uptake into synaptic vesicles in a 13

dose-dependent manner, and proposed that ATR decreases striatal DA levels, at 14

least in part, by increasing cytosolic DA, which is prone to oxidative breakdown. 15

These alterations in cellular and neurochemical targets, are likely to contribute to 16

the behavioral alterations after ATR exposure. 17

The results of this study are the first to demonstrate disruption of horizontal, 18

stereotypic, and location parameters of spontaneous locomotor activity in both 19

phases of the dark/light cycle after one year of ATR exposure. It is important to 20

note that in this study the alterations in locomotor activity were detected without the 21

need of pharmacological challenges. In contrast, a previous study used 1 mg/kg of 22

amphetamine sulfate to unmask changes in horizontal activity after two months of 23

ATR exposure [48], and hyperactivity spontaneously appeared after three months 24

21

of ATR exposure when rats reached one year of age. This observation pinpoints 1

aging as a relevant factor that renders the nigrostriatal system vulnerable. 2

An important finding of the present study is the increase in stereotypic 3

behavior (160 % compared to the control group) found in the group treated with 4

ATR for one year. Stereotypical movement is defined as a repetitive pattern of 5

movement without apparent reason and is characteristic of the obsessive 6

compulsive disorder [45]. Experimental evidence suggests that stereotypical 7

behavior could be related to alterations in the cortico-striato-thalamo-cortical loop, 8

since motor stereotypies have been induced by dopaminergic stimulation of the 9

striatum and abolished by intrastriatal blockade of dopaminergic transmission [6]. 10

The motor coordination test is used to assess the ability of an animal to 11

balance on a rotating rod, evaluating the integrity of the motor system and motor 12

skill learning. The latter is an adaptation mechanism found in humans and animals 13

important for planning and control of novel movements [5]. Both aspects of this 14

task involve the integrity of the nigrostriatal pathway, since lesions to this region 15

reduce coordination performance [38,42]. We found that the group exposed to ATR 16

maintains the same performance in all sessions, whereas the control group 17

consistently increases the time on the rotating road. Since both the ATR and 18

control groups began performing at the same level, the alterations found in this 19

task could reflect a motor skill learning deficiency. Other brain structures involved 20

in motor coordination, such as the cerebellum, could also be affected by ATR 21

exposure. The susceptibility of the cerebellum to ATR exposure was demonstrated 22

by one study where Wistar rats received a single dose of 100 mg ATR/kg, and 23

showed decrements on the spontaneous firing rate of Purkinje cells, and on 24

22

evoked cerebellar potentials, affecting mostly the response to climbing fiber input 1

[43]. 2

Exposure to 10 mg ATR/kg produced discrete alterations in the spontaneous 3

alternation task and in the non-delayed random foraging paradigm. The 4

spontaneous alternation task models the natural tendency of rodents to flexibly 5

shift between alternative spatial responses, and these behavioral strategies involve 6

different neural mechanisms associated with the prefrontal cortex [46], 7

hippocampus [57], and NAC [54]. Consequently, the lower spontaneous alternation 8

rate displayed by the ATR group, highlights possible modifications in these brain 9

structures. ATR-treated rats could be returning to the same arm as a form of 10

compulsive “checking”, or perseverative behavior in agreement with the increased 11

stereotyped behavior found during spontaneous locomotor activity, and increased 12

number of visits to previously baited arms in the non-delayed random foraging 13

paradigm. Possible effects of ATR on the NAC are suggested by the larger number 14

of errors made by rats treated with ATR in this paradigm, which is particularly 15

sensitive to the function of the NAC [19,20]. Therefore, neurotransmitter systems 16

that interact with the NAC, such as the GABAergic or glutamatergic systems, could 17

be altered by the ATR treatment, producing impairments in the performance of the 18

non-delayed foraging behavior. 19

The main effect of ATR exposure for one year on monoamine content 20

occurred in the striatum, as previously reported by others [7,48]. We found a 21

decrease of around 35% in striatal DA content in the ATR group. A smaller (13–22

15%) reduction in striatal DA content was reported previously by Rodriguez et al. 23

23

[48] using the same dose of 10 mg/ATR and a six-month exposure in Long-Evans 1

rats; the differential decrement observed in DA content could be due to the shorter 2

time of ATR exposure (six months versus one year) or to the different strains used 3

[40]. Another factor that could influence DA content is the age when exposure to 4

ATR started. In this study the herbicide exposure began at PND 21, while 5

Rodriguez et al. [48] initiated ATR treatment when rats were 9 months old. 6

It is worthwhile mentioning that endocrine disruptors widely found in the 7

environment, such as bisphenol A (BPA) and dicyclohexyl phthalate also produce 8

locomotor hyperactivity in addition to decreases in the DA transporter and in 9

tyrosine hydroxylase-positive (TH+) mesencephalic neurons in rats [29-31,36]. The 10

hyperactivity reported was also present in both phases of the dark/light cycle, as 11

observed in the present study. Although the mechanism by which exposure to BPA 12

causes hyperactivity is still unclear, it has been proposed that exposure to this 13

endocrine disruptor causes an imbalance in the function of dopamine receptors 14

within striatum, altering synaptic plasticity [58]. On the other hand, the increased 15

locomotor activity observed in rats exposed to dicyclohexyl phthalate could be 16

associated with neurodegeneration of dopaminergic cells and alterations in the 17

levels of gene expression of the D4 dopamine receptor, the dopamine transporter 18

and several subtypes of glutamate receptors in the striatum and midbrain [30]. 19

There are few reports of health-related effects of ATR exposure on human 20

populations, one study reported that increased ATR levels in drinking water have 21

been associated with intrauterine growth retardation in communities of Iowa, USA 22

[39], while augmented incidence of congenital abdominal wall defects [37] and 23

increased prevalence of small gestational age [41] were found in studies performed 24

24

in Indiana, USA. So far, ATR exposure has not been implicated in human central 1

nervous system alterations. Animal studies, such as the present one, have found 2

that exposure to this herbicide produces alterations in striatal, hypothalamic and 3

cortical monoaminergic systems [7,8,48] and behaviors associated with these 4

neuromodulator systems. Exposure to pesticides, such as rotenone, paraquat plus 5

maneb, organochlorines (dieldrin, heptachlor) has been related to alterations in the 6

dopaminergic system in rodents (for review see [32]); while in human populations 7

several reports have associated exposure to various pesticides with the 8

development of Parkinson´s disease [15-17]. Taken together, our results and 9

previous studies, suggest that ATR exposure could be a risk factor for the 10

development of neurodegenerative diseases targeting the monoaminergic 11

systems. 12

13

4. Conclusions 14

Exposure to 10 mg ATR/kg B.W. for one year disrupts spontaneous 15

locomotor activity, as shown by the alterations found in horizontal, location, and 16

stereotypic parameters both during the dark and light phases of the dark/light 17

cycle. Chronic treatment with this herbicide resulted in a significant and specific 18

decrement of DA levels in the striatum. Also, the moderate but significant 19

deficiencies found in learning tasks due to ATR exposure highlight the vulnerability 20

of other brain regions such as the prefrontal cortex and nucleus accumbens. These 21

findings suggest the nigrostriatal and the mesolimbic systems as targets of chronic 22

25

exposure to ATR, although alterations in some other neurochemical systems 1

cannot be discarded. 2

3

26

Funding 1

This work was supported by grants from CONACYT 60662 and PAPIIT 214608-19 2

to V. M. Rodríguez and CONACYT 46161-M to M. Giordano. U. Bardullas received 3

a fellowship (228586) from CONACYT. 4

5

Acknowledgments 6

We thank Dr. Dorothy Pless for her critical review of the manuscript, Fernando 7

Rodríguez, Biol. Soledad Mendoza, and MVZ. Martín García Servín for their 8

technical assistance. 9

10

Conflict of interest statement 11

The authors declare that there are no conflicts of interest regarding this research 12

article. 13

14

27

References 1

[1] J.E. Barbash, G.P. Thelin, D.W. Kolpin, R.J. Gilliom, Major herbicides in 2

ground water: results from the National Water-Quality Assessment, J 3

Environ. Qual. 30 (2001) 831-45. 4

[2] U. Bardullas, J.H. Limon-Pacheco, M. Giordano, L. Carrizales, M.S. 5

Mendoza-Trejo, V.M. Rodriguez, Chronic low-level arsenic exposure causes 6

gender-specific alterations in locomotor activity, dopaminergic systems, and 7

thioredoxin expression in mice, Toxicol. Appl. Pharmacol. 239 (2009) 169-8

77. 9

[3] M. Bentivoglio, M. Morreli, The organization and circuit of mesencephalic 10

dopaminergic neurons and the distribution of dopamine receptors in the 11

brain In: M.B. SB. Dunnett, A. M. Bentivoglio, A. Bjorklund, T. Hokfelt, 12

(Eds.), Handbook Of Chemical Neuroanatomy, Elsevier, Amsterdam, 2005, 13

pp. 62-77. 14

[4] R. Betarbet, T.B. Sherer, G. MacKenzie, M. Garcia-Osuna, A.V. Panov, J.T. 15

Greenamyre, Chronic systemic pesticide exposure reproduces features of 16

Parkinson's disease, Nat. Neurosci. 3 (2000) 1301-6. 17

[5] M.M. Buitrago, J.B. Schulz, J. Dichgans, A.R. Luft, Short and long-term 18

motor skill learning in an accelerated rotarod training paradigm, Neurobiol. 19

Learn. Mem. 81 (2004) 211-6. 20

[6] J.J. Canales, A.M. Graybiel, A measure of striatal function predicts motor 21

stereotypy, Nat. Neurosci. 3 (2000) 377-83. 22

28

[7] A. Coban, N.M. Filipov, Dopaminergic toxicity associated with oral exposure 1

to the herbicide atrazine in juvenile male C57BL/6 mice, J. Neurochem. 100 2

(2007) 1177-87. 3

[8] R.L. Cooper, S.C. Laws, P.C. Das, M.G. Narotsky, J.M. Goldman, E. Lee 4

Tyrey, T.E. Stoker, Atrazine and reproductive function: mode and 5

mechanism of action studies, Birth Defects Res. B Dev. Reprod. Toxicol. 80 6

(2007) 98-112. 7

[9] R.L. Cooper, T.E. Stoker, W.K. McElroy, J. Heien, Atrazine (ATR) disrupts 8

hypothalamic catecholamines and pituitary function [Abstract], Toxicol. Sci. 9

42 (1998) 160. 10

[10] R.L. Cooper, T.E. Stoker, L. Tyrey, J.M. Goldman, W.K. McElroy, Atrazine 11

disrupts the hypothalamic control of pituitary-ovarian function, Toxicol. Sci. 12

53 (2000) 297-307. 13

[11] B.D. Curwin, M.J. Hein, W.T. Sanderson, D.B. Barr, D. Heederik, S.J. 14

Reynolds, E.M. Ward, M.C. Alavanja, Urinary and hand wipe pesticide 15

levels among farmers and nonfarmers in Iowa, J. Expo. Anal. Environ. 16

Epidemiol. 15 (2005) 500-8. 17

[12] P.C. Das, W.K. McElroy, R.L. Cooper, Alteration of catecholamines in 18

pheochromocytoma (PC12) cells in vitro by the metabolites of chlorotriazine 19

herbicide, Toxicol. Sci. 59 (2001) 127-37. 20

[13] P.C. Das, W.K. McElroy, R.L. Cooper, Differential modulation of 21

catecholamines by chlorotriazine herbicides in pheochromocytoma (PC12) 22

cells in vitro, Toxicol. Sci. 56 (2000) 324-31. 23

29

[14] P.C. Das, W.K. McElroy, R.L. Cooper, Potential mechanisms responsible for 1

chlorotriazine-induced alterations in catecholamines in pheochromocytoma 2

(PC12) cells, Life Sci. 73 (2003) 3123-38. 3

[15] F.D. Dick, Parkinson's disease and pesticide exposures, Br. Med. Bull. 79-4

80 (2006) 219-31. 5

[16] A. Elbaz, J. Clavel, P.J. Rathouz, F. Moisan, J.P. Galanaud, B. Delemotte, 6

A. Alperovitch, C. Tzourio, Professional exposure to pesticides and 7

Parkinson disease, Ann. Neurol. 66 (2009) 494-504. 8

[17] A. Elbaz, C. Tranchant, Epidemiologic studies of environmental exposures 9

in Parkinson's disease, J. Neurol. Sci. 262 (2007) 37-44. 10

[18] N.M. Filipov, M.A. Stewart, R.L. Carr, S.C. Sistrunk, Dopaminergic toxicity of 11

the herbicide atrazine in rat striatal slices, Toxicology 232 (2007) 68-78. 12

[19] S.B. Floresco, J.K. Seamans, A.G. Phillips, A selective role for dopamine in 13

the nucleus accumbens of the rat in random foraging but not delayed spatial 14

win-shift-based foraging, Behav. Brain Res. 80 (1996) 161-8. 15

[20] S.B. Floresco, J.K. Seamans, A.G. Phillips, Selective roles for hippocampal, 16

prefrontal cortical, and ventral striatal circuits in radial-arm maze tasks with 17

or without a delay, J. Neurosci. 17 (1997) 1880-90. 18

[21] S.B. Floresco, J.R. St Onge, S. Ghods-Sharifi, C.A. Winstanley, Cortico-19

limbic-striatal circuits subserving different forms of cost-benefit decision 20

making, Cogn. Affect. Behav. Neurosci. 8 (2008) 375-89. 21

[22] A.S. Friedmann, Atrazine inhibition of testosterone production in rat males 22

following peripubertal exposure, Reprod. Toxicol. 16 (2002) 275-9. 23

30

[23] D.W. Gammon, C.N. Aldous, W.C. Carr, Jr., J.R. Sanborn, K.F. Pfeifer, A 1

risk assessment of atrazine use in California: human health and ecological 2

aspects, Pest. Manag. Sci. 61 (2005) 331-55. 3

[24] G. Giusi, R.M. Facciolo, M. Canonaco, E. Alleva, V. Belloni, F. Dessi'-4

Fulgheri, D. Santucci, The endocrine disruptor atrazine accounts for a 5

dimorphic somatostatinergic neuronal expression pattern in mice, Toxicol. 6

Sci. 89 (2006) 257-64. 7

[25] J.A. Grahn, J.A. Parkinson, A.M. Owen, The role of the basal ganglia in 8

learning and memory: neuropsychological studies, Behav. Brain Res. 199 9

(2009) 53-60. 10

[26] H. Gysin, E. Knuesli, Chemistry and herbicidal properties of triazine 11

derivatives In: R. Metcalf (Ed.), Advances in pest control research, 12

Interscience, New York, 1960, pp. 289-358. 13

[27] T.B. Hayes, A. Collins, M. Lee, M. Mendoza, N. Noriega, A.A. Stuart, A. 14

Vonk, Hermaphroditic, demasculinized frogs after exposure to the herbicide 15

atrazine at low ecologically relevant doses, Proc. Natl. Acad. Sci. U S A. 99 16

(2002) 5476-80. 17

[28] M.M. Hossain, N.M. Filipov, Alteration of dopamine uptake into rat striatal 18

vesicles and synaptosomes caused by an in vitro exposure to atrazine and 19

some of its metabolites, Toxicology 248 (2008) 52-8. 20

[29] M. Ishido, Y. Masuo, M. Kunimoto, S. Oka, M. Morita, Bisphenol A causes 21

hyperactivity in the rat concomitantly with impairment of tyrosine 22

hydroxylase immunoreactivity, J. Neurosci. Res. 76 (2004) 423-33. 23

31

[30] M. Ishido, Y. Masuo, J. Sayato-Suzuki, S. Oka, E. Niki, M. Morita, 1

Dicyclohexylphthalate causes hyperactivity in the rat concomitantly with 2

impairment of tyrosine hydroxylase immunoreactivity, J. Neurochem. 91 3

(2004) 69-76. 4

[31] M. Ishido, J. Yonemoto, M. Morita, Mesencephalic neurodegeneration in the 5

orally administered bisphenol A-caused hyperactive rats, Toxicol. Lett. 173 6

(2007) 66-72. 7

[32] D.C. Jones, G.W. Miller, The effects of environmental neurotoxicants on the 8

dopaminergic system: A possible role in drug addiction, Biochem. 9

Pharmacol. 76 (2008) 569-81. 10

[33] S.C. Laws, M. Hotchkiss, J. Ferrell, S. Jayaraman, L. Mills, W. Modic, N. 11

Tinfo, M. Fraites, T. Stoker, R. Cooper, Chlorotriazine herbicides and 12

metabolites activate an ACTH-dependent release of corticosterone in male 13

Wistar rats, Toxicol. Sci. 112 (2009) 78-87. 14

[34] S. Lim, S.Y. Ahn, I.C. Song, M.H. Chung, H.C. Jang, K.S. Park, K.U. Lee, 15

Y.K. Pak, H.K. Lee, Chronic exposure to the herbicide, atrazine, causes 16

mitochondrial dysfunction and insulin resistance, PLoS One 4 (2009) e5186. 17

[35] P.J. Lioy, R.D. Edwards, N. Freeman, S. Gurunathan, E.D. Pellizzari, J.L. 18

Adgate, J.J. Quackenboss, K. Sexton, House dust levels of selected 19

insecticides and a herbicide measured by the EL and LWW samplers and 20

comparisons to hand rinses and urine metabolites, J. Expo. Anal. Environ. 21

Epidemiol. 10 (2000) 327-40. 22

[36] Y. Masuo, M. Morita, S. Oka, M. Ishido, Motor hyperactivity caused by a 23

deficit in dopaminergic neurons and the effects of endocrine disruptors: a 24

32

study inspired by the physiological roles of PACAP in the brain, Regul. Pept. 1

123 (2004) 225-34. 2

[37] K.D. Mattix, P.D. Winchester, L.R. Scherer, Incidence of abdominal wall 3

defects is related to surface water atrazine and nitrate levels, J. Pediatr. 4

Surg. 42 (2007) 947-59. 5

[38] C. Monville, E.M. Torres, S.B. Dunnett, Comparison of incremental and 6

accelerating protocols of the rotarod test for the assessment of motor 7

deficits in the 6-OHDA model, J. Neurosci. Methods 158 (2006) 219-23. 8

[39] R. Munger, P. Isacson, S. Hu, T. Burns, J. Hanson, C.F. Lynch, K. 9

Cherryholmes, P. Van Dorpe, W.J. Hausler, Jr., Intrauterine growth 10

retardation in Iowa communities with herbicide-contaminated drinking water 11

supplies, Environ. Health Perspect. 105 (1997) 308-14. 12

[40] M.G. Narotsky, D.S. Best, D.L. Guidici, R.L. Cooper, Strain comparisons of 13

atrazine-induced pregnancy loss in the rat, Reprod. Toxicol. 15 (2001) 61-9. 14

[41] H. Ochoa-Acuna, J. Frankenberger, L. Hahn, C. Carbajo, Drinking-water 15

herbicide exposure in Indiana and prevalence of small-for-gestational-age 16

and preterm delivery, Environ. Health Perspect. 117 (2009) 1619-24. 17

[42] E.Y. Pioli, W. Meissner, R. Sohr, C.E. Gross, E. Bezard, B.H. Bioulac, 18

Differential behavioral effects of partial bilateral lesions of ventral tegmental 19

area or substantia nigra pars compacta in rats, Neuroscience 153 (2008) 20

1213-24. 21

[43] M.V. Podda, F. Deriu, A. Solinas, M.P. Demontis, M.V. Varoni, A. Spissu, V. 22

Anania, E. Tolu, Effect of atrazine administration on spontaneous and 23

evoked cerebellar activity in the rat, Pharmacol. Res. 36 (1997) 199-202. 24

33

[44] J.L. Rayner, R.R. Enoch, D.C. Wolf, S.E. Fenton, Atrazine-induced 1

reproductive tract alterations after transplacental and/or lactational exposure 2

in male Long-Evans rats, Toxicol. Appl. Pharmacol. 218 (2007) 238-48. 3

[45] R.M. Ridley, The psychology of perserverative and stereotyped behaviour, 4

Prog. Neurobiol. 44 (1994) 221-31. 5

[46] T.W. Robbins, Chemistry of the mind: neurochemical modulation of 6

prefrontal cortical function, J. Comp. Neurol. 493 (2005) 140-6. 7

[47] V.M. Rodriguez, L. Carrizales, M.E. Jimenez-Capdeville, L. Dufour, M. 8

Giordano, The effects of sodium arsenite exposure on behavioral 9

parameters in the rat, Brain Res. Bull. 55 (2001) 301-8. 10

[48] V.M. Rodriguez, M. Thiruchelvam, D.A. Cory-Slechta, Sustained exposure 11

to the widely used herbicide atrazine: altered function and loss of neurons in 12

brain monoamine systems, Environ. Health Perspect. 113 (2005) 708-15. 13

[49] M.K. Ross, T.L. Jones, N.M. Filipov, Disposition of the Herbicide 2-Chloro-4-14

(ethylamino)-6-(isopropylamino)-s-triazine (Atrazine) and Its Major 15

Metabolites in Mice: A Liquid Chromatography/Mass Spectrometry Analysis 16

of Urine, Plasma, and Tissue Levels, Drug Metab. Dispos. 37 (2009) 776-17

86. 18

[50] W. Schultz, Multiple dopamine functions at different time courses, Annu. 19

Rev. Neurosci. 30 (2007) 259-88. 20

[51] T.J. Shafer, T.R. Ward, C.A. Meacham, R.L. Cooper, Effects of the 21

chlorotriazine herbicide, cyanazine, on GABA(A) receptors in cortical tissue 22

from rat brain, Toxicology 142 (1999) 57-68. 23

34

[52] T.E. Stoker, R.L. Cooper, Distribution of (14)C-atrazine following an acute 1

lactational exposure in the Wistar rat, Reprod. Toxicol. 23 (2007) 607-10. 2

[53] T.E. Stoker, C.L. Robinette, R.L. Cooper, Maternal exposure to atrazine 3

during lactation suppresses suckling- induced prolactin release and results 4

in prostatitis in the adult offspring, Toxicol. Sci. 52 (1999) 68-79. 5

[54] K. Taghzouti, A. Louilot, J.P. Herman, M. Le Moal, H. Simon, Alternation 6

behavior, spatial discrimination, and reversal disturbances following 6-7

hydroxydopamine lesions in the nucleus accumbens of the rat, Behav. 8

Neural Biol. 44 (1985) 354-63. 9

[55] M. Thiruchelvam, E.K. Richfield, R.B. Baggs, A.W. Tank, D.A. Cory-Slechta, 10

The nigrostriatal dopaminergic system as a preferential target of repeated 11

exposures to combined paraquat and maneb: implications for Parkinson's 12

disease, J. Neurosci. 20 (2000) 9207-14. 13

[56] G. Ugazio, A. Bosio, E. Burdino, L. Ghigo, C. Nebbia, Lethality, hexobarbital 14

narcosis and behavior in rats exposed to atrazine, bentazon or molinate, 15

Res. Commun. Chem. Pathol. Pharmacol. 74 (1991) 349-61. 16

[57] S.R. White, Atropine, scopolamine and hippocampal lesion effects on 17

alternation performance of rats, Pharmacol. Biochem. Behav. 2 (1974) 297-18

307. 19

[58] R. Zhou, Z. Zhang, Y. Zhu, L. Chen, M. Sokabe, Deficits in development of 20

synaptic plasticity in rat dorsal striatum following prenatal and neonatal 21

exposure to low-dose bisphenol A, Neuroscience 159 (2009) 161-71. 22

23

24

25

35

Figure legends 1

Fig. 1. Experimental design, and timeline events for chronic ATR exposure during 2

one year. 3

4

Fig. 2. Locomotor activity in ATR-exposed animals as percent of control during the 5

first hour in the spontaneous locomotor activity chambers evaluated over 12 6

months of exposure. Each bar in the graph represents the mean (± SEM). 7

* Different from control group, p < 0.05 with Bonferroni correction. 8

9

Fig. 3. Spontaneous locomotor activity evaluated for 24 hours during the dark (A-10

C) and light phase (A’-C’) of the dark/light cycle at eight (A, A’), ten (B, B’), or 11

twelve months (C, C’) of ATR exposure. Vertical bars indicate the percent 12

difference between the group exposed to 10 mg ATR/kg and the control group. 13

Abbreviations: HA, horizontal activity; TD, total distance; MN, number of 14

movements; MT, movement time; VA, vertical activity; VT, vertical time; SC, 15

stereotypy counts; SN, number of stereotypy; ST, stereotypy time; MD, margin 16

distance; CD, center distance. 17

* Different from control group, p < 0.05 with Bonferroni correction. 18

19

Fig. 4. Horizontal activity (A) and total distance (B) of exposed and control rats 20

recorded over the course of a 24-h dark/light cycle after twelve months of ATR 21

exposure. Horizontal activity and total distance increased during the dark [t´s (15) = 22

36

3.308 – 3.96, p = 0.002 - 0.0003] and light phases of the cycle [t´s (15) = 3.241 – 1

4.603, p = 0.004 - 0.001]. 2

3

Fig. 5. Effect of the ATR exposure on motor coordination. Exposure to 10 mg 4

ATR/kg diminished the time on the cylinder in comparison to the control group [t´s 5

(12) = - 2.249, - 2.334, p = 0.044 – 0.037]. Each symbol represents the mean of 6

latency and revolutions per second to fall off the rotating cylinder. 7

8

Fig. 6. Effect of chronic ATR exposure on the performance of a non-delayed 9

random foraging task. Exposure to 10 mg ATR/kg increased the number of re-10

entry errors in baited arms. 11

* Different from control group, p = 0.0300; ~ p = 0.0652. 12

13

Fig. 7. These graphs show the linear relationship between various measures of 14

locomotor activity and dopamine levels in significance level, are shown. Only for 15

the striatum was the regression analysis significant, and only dopamine was a 16

significant predictor of locomotor activity (see text).the striatum (ng/mg protein). In 17

each graph the correlation coefficient, as well the 18

19

20

21

22

Comentario [MG2]: Se cortó este pie de figura, es necesario completarlo.