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Background:
Organophosphate compounds represent a major class of chemicals commonly applied in agricultural pest control [5]. Most studies of moderate pesticide exposure have found an increased prevalence of neurological symptoms and changes in neurobehavioral performance, which reflects cognitive and psychomotor dysfunction [2]. Glyphosate (GLY) is the active ingredient of the most used herbicide worldwide [1]. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38- amino-acid neuropeptide that belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP) [3]; [4]. Several in vitro and in vivo studies revealed that PACAP exerts its neuroprotective properties. Anxiety and depression are highly co-morbid with each other. This research aims to study the toxicity of GLY and the effect of the neuropeptide PACAP, accompanied by behavioral studies to test anxiety and depression.

Materials/Methods:
In vivo, a sexually mature male Swiss albino mice model with repeated exposure to GLY and/or PACAP for 15 days elicited evident emotional behavioral alterations as assessed by several behavioral tests after 48 hours of stopping treatment. Four tests were performed. The elevated-plus-maze test is used to assess anxiety-like behavior in rodents as well as the open field test to further assess locomotor activity and emotional reactivity. The tail suspension and the dark/light box tests are used to evaluate potential antidepressant-like effects. The apparatus was cleaned with a solution of ethanol of 10 % between tests to remove animal odors. Time was recorded using a video camera and analyzed by the Ethovision XT Noldus 8.5 video tracking program. All results were analyzed using GraphPad Prism 8.0 (GraphPad Software Inc.). The data are represented as the mean ± standard deviation (SD) of at least three independent experiments when shown as a bar graph. Statistical comparisons were performed between the control and treatment groups using Tukey's post hoc test for multiple comparisons of one-way analysis of variance. p < 0.05 indicated significantly difference (*); P<0.01 difference is extremely significant (**) and p<0.001 difference is highly significant (***).

Results:
Repeated exposure to GLY and/or PACAP for 15 days elicited evident emotional behavioral alterations in mice, as assessed first by the Tail Suspension Test (TST) in which the total time spent immobile during the last 4 minutes of a session was scored to evaluate potential antidepressant-like effects in mice. The statistical analysis of the immobility time recorded of the TST showed that the glyphosate group presented an increase in the percentage of immobility time compared to the control indicating an elevation of depressive-like behavior in that group (highly significant **). However, compared to the glyphosate group, the co-treated group demonstrated a decrease in immobility time (compared to the Glyphosate group) illustrating the effects of PACAP in alleviating depressive-like behavior. Second, for the dark/light box test in two-chambered systems, where animals can freely move between brightly lit open fields and dark corners, mice show more crossings between two chambers and more locomotor activity after treatment with GLY. The time spent in the bright compartment was an extremely significant (****) increase for the two treatments with the herbicide and with the neuropeptide compared to control groups and an extremely significant (####) decrease in the PACAP group compared to the Glyphosate group. We recovered 11 boli of mice feces and urine from the herbicide-treated group, eight boli for the PACAP group seven boli from the co-treated groups, and nothing from the control group. Then, for the elevated-plus-maze test, the time spent in open arms is used as indices of open space-induced anxiety. The percent of entries and time spent in each arm constitute the index of primary anxiety. The analysis of the ratio of open arm (OA) entries showed a slight decrease in several entries compared to the control group, which is statistically insignificant. Similarly, the glyphosate group spent less time exploring the OA. On the other hand, the GLY + PACAP group spent a moderate amount of time exploring the OA. We can deduce that anxious mice spend little time in the open arms and make very few entries into the open arms. The anxiety index showed that treatment with PACAP relieves depression behavior. Ultimately, we performed the Open field test which consists of placing an animal in an unknown environment with surrounding walls, to observe behavior patterns including: ? The tendency to stay on the periphery of the field without entering the center (called thigmotaxis) and often interpreted as anxious behavior. ? Level of defecation and urination, in which I recovered 10 boli of feces for the group treated with GLY and 7 boli for the co-treated groups, and nothing recovered for the control and PACAP-treated groups. Therefore, the time spent in the center area is an index of anxiety behavior, this test assesses locomotor activity and emotional reactivity.

Conclusion:
With the widespread use of pesticides, the impact of pesticides on host health has received more and more attention. Glyphosate exposure induced an exacerbation of anxiety and depression-like behaviors and treatment with PACAP alleviate it. Our findings provide strong evidence that the neuropeptide PACAP can be used as a novel treatment option for neurological disorders and provide novel insights into the mechanism underlying glyphosate-induced health risks, particularly mood disorders.

References:

References:

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