Research reportAPOE genetic background and sex confer different vulnerabilities to postnatal chlorpyrifos exposure and modulate the response to cholinergic drugs
Introduction
The impact of environmental toxic exposure during brain development has enormous consequences for the general population. As far as subclinical behavioral or cognitive deficits caused by toxic exposure are elusive in diagnoses, most of the neurotoxic agents affecting the brain lack specific environmental regulations. This is the case of the worldwide used pesticide chlorpyrifos (CPF), which targets the cholinergic neurotransmitter system. Despite the fact that its residential use was banned in 2001, CPF is still one of the most widespread compounds. The general population is permanently exposed to nontoxic low levels of the pesticide through the diet [1], being known that young subjects are more sensitive than adults to CPF toxicity [2,3]. The main detrimental effects of CPF are caused by the irreversible inhibition of cholinesterase enzymes (ChE) such as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) [4]. To date, a number of studies have reported changes in the cholinergic system after exposure to CPF. For instance, Slotkin et al. [5] found a decreased binding in both α7 and α4β2 nicotinic receptors (nAChRs) in different brain regions of rats including the forebrain and cerebellum, following postnatal (postnatal days (PND) 11–14) exposure to the pesticide. In the same line, Richardson and Chambers [6] observed a considerable dose-dependent decrease in muscarinic receptors (mAChRs) binding in the brain after postnatal (PND 1–21) exposure to CPF. The observed effects, which cover a wide spectrum, seem to be dependent on the sex and the period of exposure. For example, postnatal exposure to CPF has been related to increased aggressive behavior in adult males and an enhanced maternal response in females, while anxiety-like behaviors have been reduced in both sexes [7]. Likewise, postnatal exposure to CPF is known to induce changes in locomotor activity [8,9], as well as thyroid alterations [10].
To date, a number of studies have focused on the gene of apolipoprotein E (apoE) as another potential factor underlying cholinergic alterations. The apoE protein is implicated in lipid and cholesterol transport in the brain [11]. Three different isoforms have been described in humans: apoE2, apoE3 and apoE4. E4 is the isoform related to diminished functioning of the cholinergic system [12], being the major genetic risk factor for the development of Alzheimer’s Disease (AD) [13,14]. Previous results from our group, using the targeted replacement (TR) mouse model that expresses the different human isoforms of apoE (apoE-TR), showed a wide variety of functional differences between genotypes as well as different vulnerabilities to the detrimental effects of CPF exposure. There are differences between genotypes, for example, in terms of spatial learning and memory [15], attention, impulsivity and compulsivity [16,17], and these influence the effects of CPF during adulthood [18] or throughout the developmental period [19]. Likewise, differences in the gene expression of cholinergic elements were observed between genotypes after postnatal exposure to the pesticide, together with developmental alterations [20].
Behavioral testing has played a crucial role in evaluating the effects of toxic exposures and in unraveling their interactions with environmental factors. The Object Recognition Test (ORT) is a behavioral task used to study recognition memory, which is based on the innate preference of rodents for novelty (i.e., to explore a new object over a familiar one). This innate novelty preference is ultimately used to test recognition memory because mice preferring a novel object would need to successfully recognize a familiar one [21]. In mice, the brain structures likely to be implicated in object recognition memory are the hippocampus and rhinal cortices. More specifically, the perirhinal cortex is a crucial structure in the acquisition, consolidation and retrieval of information [[22], [23], [24]]. The use of ORT has increased in recent years because of advantages over other well-established tasks: it does not require water or food deprivation, reinforcing stimuli or the learning of associations, being suitable for pharmacological screening [25,26]. The integrity of the cholinergic system appeared to be determinant for correct recognition memory. The use of the cholinergic antagonist scopolamine has become a widely established pharmacological model for cognitive impairments on object recognition [27,28]. On the other hand, the use of nAChR agonists and AChE inhibitors [29,30] has been associated with an improvement in the performance of the task. The GABAergic system has also been reported to play a role in recognition memory as the administration of γ-Aminobutyric acid (GABA) induced long-term improvements in recognition memory in rats [31]. Amnesic effects and subsequent impairments have also been described in recognition memory after administration of an allosteric modulator of GABAA receptor such as alprazolam [32,33].
The purpose of this investigation was to assess the effects of postnatal exposure to CPF on recognition memory and to investigate how individual intrinsic variables including sex and APOE genotype influence its toxicity. We also aimed to shed light on the neuropharmacological basis of these effects. To explore the role of both cholinergic and GABAergic systems on recognition memory and their interactions with the APOE genotype and CPF, a pharmacological challenge with agonist and antagonist drugs was used.
Section snippets
Animals and care
In the present study, we used male and female apoE3- and apoE4-TR mice from Taconic (Taconic Europe, Lille Skensved, Denmark) and C57BL/6 mice from Charles River (Charles River, Barcelona, Spain). The apoE-TR mouse model was originally created by Sullivan et al. [34]. These mice have a C57BL/6NTac background and their murine apoE gene was replaced by the human allele APOE. Females and males from the same genotype were subjected to mating sessions up to pregnancy. The day at birth was recorded
General activity during habituation
General activity during habituation was analyzed by a three-way ANOVA (sex x genotype x treatment) for repeated measures. The 5-minute fractions of time were the within-subject factor and the distance traveled as well as the time spent in the center of the open field were studied as the dependent variables. A decrease over time in the total distance traveled [F(5,129) = 69.268, p < 0.001] indicated habituation to the space (Fig. 1A and B). An interaction between time and genotype
Discussion
The current investigation was aimed at studying in mice the effects of postnatal exposure to the pesticide CPF on recognition memory. An ORT was used to assess recognition memory, based on the premise that preference for a novel object means that the representation of the familiar object still exists in the memory of the animals. The potential implication of both the GABAergic and the cholinergic systems was assessed according to sex, APOE genotype and postnatal treatment using pharmacological
Acknowledgements
The authors would like to thank Dr Celeste di Paolo, Esperanza Chernichero and Juan Valencia for their technical support with animal care. This research was supported by the Spanish Ministry of the Economy and Competitiveness (MINECO, Spain) (Grant Numbers PSI2014-55785-C2-2-R; PSI2014- 55785-C2-1-R; PSI2017-86847-C2-2-R).
References (69)
- et al.
Age- and gender-related differences in the time course of behavioral and biochemical effects produced by oral chlorpyrifos in rats
Toxicol. Appl. Pharmacol.
(1998) - et al.
Comparison of in vivo cholinesterase inhibition in neonatal and adult rats by three organophosphorothioate insecticides
Toxicology.
(1991) - et al.
α7 Nicotinic acetylcholine receptors targeted by cholinergic developmental neurotoxicants: nicotine and chlorpyrifos
Brain Res. Bull.
(2004) - et al.
Developmental exposure to chlorpyrifos alters reactivity to environmental and social cues in adolescent mice
Toxicol. Appl. Pharmacol.
(2003) - et al.
Chlorpyrifos exposure during a critical neonatal period elicits gender-selective deficits in the development of coordination skills and locomotor activity
Dev. Brain Res.
(2000) - et al.
Apoprotein E as a lipid transport and signaling protein in the blood, liver, and artery wall
J. Lipid Res.
(2009) - et al.
Reduced cholinergic function in normal and Alzheimer’s disease brain is associated with apolipoprotein E4 genotype
Neurosci. Lett.
(1997) Brain lipid metabolism, apolipoprotein E and the pathophysiology of Alzheimer’s disease
Neuropharmacology.
(2010)- et al.
Behavioral phenotype and BDNF differences related to apoE isoforms and sex in young transgenic mice
Exp. Neurol.
(2012) - et al.
Apolipoprotein E (APOE) genotype and the pesticide chlorpyrifos modulate attention, motivation and impulsivity in female mice in the 5-choice serial reaction time task
Food Chem. Toxicol.
(2016)