Behavioural and Neurochemical characterization of the effects of Fipronil in the rat brain

The aim of my thesis is to better characterize the neurotoxic effect of fipronil (FPN), a phenylpyrazole pesticide which is used to prevent insects such as fleas and ticks from plaguing cats, dogs and other pets as well as in agriculture for repelling a variety of insects from crops and homes (termites, fire ants, etc.). For this purpose, two types of experiments were performed in adult male rats: the first experiment (Part 1 of the thesis) was aimed at studying the effect of FPN injected unilaterally, directly into the right substantia nigra (SN) of male rats on different tests (i.e., open field, rotarod test, tail flick test, novel object recognition test, social interaction test) 7 and 14 days after the injection, accordingly, to study that a deficit in the performance in these tests well correlate to a degeneration of the nigrostiatal dopaminergic system. Rats unilaterally injected with vehicle (DMSO) into right SN were used as controls. Unilaterally SN FPN-treated and control rats were also treated with a systemic challenge dose of the DA-agonist apomorphine to study the presence of a rotational behaviour, possibly correlated to a degeneration of the nigrostriatal dopaminergic system. Fifteen days after the injection, right SN FPN-treated and SN control DMSO-rats were sacrificed, their striata was removed for the determination of DA content in the ipsilateral and in the intact controlateral striatum as well as the SN was used for tyrosine hydroxylase (TH) immunoreactivity (by immunohistochemistry) in order to correlate behavioral changes to the neurodegeneration of the nigrostriatal dopaminergic system. The results confirm that FPN unilateral injection into the right SN caused a degeneration of the nigrostiatal dopaminergic neurons which leads to a decrease around 50% in striatal DA content and SN TH imunoreactivity with respect to control values. These changes are usually, but not always, correlated with changes in motor activity and coordination, in nociception and cognition, and resemble those found in rats treated with other neurotoxins in the SN (6-hydroxydopamine, rotenone, MPTP and MPP+). The second type of experiment (part 2 of the thesis) was aimed at studing the effect of FPN chronically administered by oral gavage for 21 days to adult male rats on brain monoaminergic systems in order to evaluate possible alteration in the content of monoamines [noradrenaline (NA), DA, serotonin (5-HT) and their metabolites] and thus on monoaminergic connectivity across different brain areas induced by the exposure to the pesticide. FPN was able to significantly decrease DA and its metabolites levels in most striatal territories including the core of the nucleus accumbens and the substantia nigra (SN). The pesticide also diminished 5-HT levels in some striatal regions including the core, anterior and ventral striatum, and in SN. The indirect index of the turnover 3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratio and 5-hydroxyindole-3-acetic acid (5-HIAA)/5-HT ratio were correspondingly increased in numerous brain regions. FPN reduced NA content only in the core of the nucleus accumbens. Using the Bravais-Pearson test to study the neurochemical organization of monoamines through multiple correlative analyses across the brain, fewer correlations for NA, DOPAC/DA ratio and 5-HIAA/5-HT ratio, and an altered pattern of correlations within and between monoamine systems were found. This suggests that chronic exposure to FPN quantitatively reduces DA and 5-HT content in some brain regions and qualitatively leads to changes in the whole central monoamine connectivity. Together, the results of my thesis confirm that FPN exerts neurotoxic effects in the rat brain, that are accompanied by a decrease in DA and 5-HT content in the striatal territory, supporting the hypothesis that exposure to this pesticide may also contribute to the initiation and/or progression of Parkinson’s disease in humans.