Chlorpyrifos exposure during neurulation: cholinergic synaptic dysfunction and cellular alterations in brain regions at adolescence and adulthood

Abstract

The developmental neurotoxicity of chlorpyrifos (CPF) involves multiple mechanisms, thus rendering the immature brain susceptible to adverse effects over a wide window of vulnerability. Earlier work indicated that CPF exposure at the neural tube stage elicits apoptosis and disrupts mitotic patterns in the brain primordium but that rapid recovery ensues before birth. In the current study, we assessed whether defects in cholinergic synaptic activity emerge later in development. CPF was given to pregnant rats on gestational days 9–12, using regimens devoid of overt maternal or fetal toxicity. We then examined subsequent development of acetylcholine systems and compared the effects to those on general biomarkers of cell development. Choline acetyltransferase (ChAT), a constitutive marker for cholinergic nerve terminals, was increased in the hippocampus and striatum in adolescence and adulthood. In contrast, hemicholinium-3 (HC-3) binding to the presynaptic choline transporter, an index of nerve impulse activity, was markedly subnormal. Furthermore, m₂-muscarinic cholinergic receptor binding was significantly reduced, instead of showing the expected compensatory upregulation for reduced neural input. CPF also elicited delayed-onset alterations in biomarkers of cell packing density, cell number, cell size and neuritic projections, involving brain regions both with and without reductions in indices of cholinergic activity. In combination with earlier results, the current findings indicate that the developing brain, and especially the hippocampus, is adversely affected by CPF regardless of whether exposure occurs early or late in brain development, and that defects emerge in adolescence or adulthood even in situations where normative values are initially restored in the immediate post-exposure period.

Introduction

It is increasingly evident that the developmental neurotoxicities of chlorpyrifos (CPF) and other organophosphate insecticides depend on a variety of mechanisms, rather than reflecting simply the inhibition of cholinesterase [2], [32], [36], [42]. Accordingly, their impact is evident over a wide developmental period, comprising neural cell proliferation and differentiation, axonogenesis and synaptogenesis, and ultimately, synaptic function [2], [20], [21], [29], [32], [33], [36], [42]. Indeed, adverse effects are exerted toward glia as well as neurons, so that vulnerability continues well into the postnatal period [2], [11], [12], [13], [28], [34].

In a recent series of studies, we compared biochemical indices of brain cell damage in developing rats exposed to CPF during late gestation or postnatally [12], [13], [33], [42] and found that postnatal exposure had a greater, immediate effect on the number of brain cells and on general indices of fetal synaptic development. However, when we examined more selective targeting of cholinergic systems, there was evidence for specific disruption with late gestational exposure, even at CPF doses below the threshold for fetal growth impairment or for inhibition of fetal brain cholinesterase [33]. When these animals were examined in the early postnatal period and into adolescence and adulthood, they showed later-emerging, persistent deficits in cholinergic synaptic function and related cognitive behavioral performance [23], [35]. Interestingly, although there was evidence for delayed neurotoxicity (reductions in cell number and packing density and subnormal indices of neuritic outgrowth), the cholinergic deficits did not correlate temporally or regionally with the cellular toxicity markers [35]. This suggests that cholinergic projections are selectively targeted for delayed alterations evoked by prenatal CPF exposure, over and above more general effects on neural cell development.

The current study takes a similar approach to the mechanisms underlying the anomalies associated with much earlier prenatal CPF exposure, during the period of neurulation, addressing three specific questions. First, does CPF exposure as early as neurulation cause a delayed deficit in cholinergic synaptic function? Second, are these alterations related to generalized defects in brain cell development, or are they specific to cholinergic systems? Third, is there any regional selectivity to the effects, given that the exposure occurs during the earliest stages of brain formation? Studies in embryo culture at the neural tube stage indicate widespread apoptosis, interference with mitosis and abnormal architecture in the brain primordium after even a brief period of CPF exposure [38], yet when biomarkers of neural cell development are examined several days later, there is only minor evidence of neurotoxicity [33]. Given the later emergence of cholinergic dysfunction in our earlier study of late gestational exposure [35], we now extend these results to CPF administered in the neural tube stage, examining effects in adolescence and adulthood.

For cholinergic synaptic development, we assessed choline acetyltransferase activity (ChAT), an archetypal measure of cholinergic innervation [1], [14], [31], [44], [51], [52], as well as the binding of [³H]hemicholinium-3 (HC-3) to the presynaptic high-affinity choline transporter, which is responsive to neuronal activity [18], [41]. The comparative changes in ChAT and HC-3 binding thus permit distinction between effects on synaptic outgrowth as distinct from synaptic activity [1], [14], [31], [44], [51], [52], and have been well-characterized for effects of CPF on cholinergic systems in adult rats [26], [27] and for immediate and delayed effects of CPF exposure during other developmental periods [8], [33], [35], [43]. We also measured radioligand binding to the m₂-muscarinic acetylcholine receptor (m₂AChR), a mediator of cholinergic signaling that typically undergoes downregulation in the presence of cholinergic hyperstimulation [4], [5], [49] and that may also be a direct target for CPF actions [3], [15]. Measurements of DNA and cell protein fractions were used to evaluate CPF's general effects on cell development: DNA content (DNA per brain region) reflects the total number of cells, DNA concentration (DNA per unit tissue weight), reflects the cell packing density, total protein/DNA reflects relative cell size and membrane protein/total protein reflects the development of neuritic projections. The rationale and mechanistic basis for each of these markers was presented in our earlier studies [33], [35].