Excitation/Inhibition Dysregulation of Neuronal Development
Rubenstein and Merzenich (2003) elegantly described a model of autism whereby the cortical networks that govern language and social behavior are skewed toward increased excitation (i.e., away from inhibition), resulting in an overall hyperexcitable state. Their hypothesis addresses both genomic and environmental factors influencing glutamate and GABA (γ-aminobutyric acid)–mediated neurotransmission, resulting in more noise in neural networks.
By poundage applied, the majority of pesticides inhibit AChE. Examples include two of the most frequently used pesticides worldwide, the OPs chlorpyrifos and diazinon, as well as the monomethyl carbamates (CBs), including propoxur and methomyl. Insecticides that target voltage-gated sodium channels (e.g., pyrethroids and DDT), the nicotinic ACh receptors (nAChR) themselves (e.g., imidacloprid), and GABAA receptors (e.g., OC and fipronil) are ranked next highest in use in overall poundage applied (Casida 2009). The levels of ACh- and GABA-mediated neurotransmission and the activity of voltage-dependent sodium channels are critical throughout prenatal and postnatal development, defining the ratio of excitatory and inhibitory neurotransmission in the brain, but also promoting and refining neural networks in the developing and adult brain (Belmonte and Bourgeron 2006).
GABA Signaling Pathways
GABA is critical for normal development and regulation of neurotransmission (Campbell 1996). GABA activates two major families of receptors expressed in the mammalian brain: a) GABAA and GABAC receptors that promote chloride fluxes, and b) GABAB receptors that are coupled to G-protein signaling. In adults, GABAA receptor activation promotes chloride influx and hyperpolarization of the membrane, decreasing neuronal excitability. However, during fetal development, the chloride gradients across the membrane are reversed, and therefore activation of GABAA receptors in the hippocampus and neocortex causes net chloride efflux and enhanced excitation (Watanabe et al. 2002). Thus, the temporal expression and spatial localization of GABA receptors within the brain can determine the patterns and activity of neural circuits. Numerous subunit isoforms for the GABAA receptor are developmentally regulated during the perinatal period and have distinct biophysical and pharmacological properties that contribute to their physiological (Cossart et al. 2005) and pathophysiological (Stafstrom et al. 2010) functions. GABA is known to regulate many aspects of neural stem cell proliferation, differentiation, migration, and elongation (Varju et al. 2001). Because of observed deficits in social and exploratory behavior, the GABAA receptor β3-gene–deficient mouse has been suggested as an animal model of autism spectrum disorder (DeLorey et al. 2008).
Disruptions in the GABA system have been reported to be associated with autism in studies of receptor density from brain tissue (Blatt et al. 2001) as well as in studies of genetic association (Buxbaum et al. 2002; Cook et al. 1998; McCauley et al. 2004). In postmortem cerebellar tissue samples from the brains of adults with autism, relative numbers of GABAA receptors were reduced in 4 cases as compared with 8 controls, and GABAB expression was altered in 5 cases as compared with 7 controls (Fatemi et al. 2009a, 2009b). Decreased expression of GABAA receptor β3 was shown to be associated with MECP2 (the gene for methyl CpG binding protein 2) impairment in brain tissue samples from cases of autism, Angelman syndrome, and Rett syndrome (Samaco et al. 2005). In a family-based study, single nucleotide polymorphisms were examined in 470 families with at least one case of autism (266 multiplex, 204 triads) for GABA subunits on 14 alleles. Findings showed significant associations for GABAA receptor polymorphisms, in particular the A4 subunit and gene × gene interaction between receptor subunits (Ma et al. 2005).
In rats, prenatal exposure to the OC pesticides dieldrin and lindane reduces GABAA receptor binding capabilities in the brainstem (Brannen et al. 1998). In another rat study, prenatal dieldrin exposure was found to alter mRNA expression and subunit composition of GABAA receptors (Liu et al. 1998). Results from in vitro cortical neuronal cultures have shown endosulfan and related OC pesticides to increase Akt phosphorylation, an effect mediated by the activation of ERβ, and to activate ERK1/2 through a mechanism involving GABAA and glutamate receptors (Briz et al. 2011). In humans, a diminished ability to bind GABA contributes to poor muscle tone, which is observed in over half of persons with autism (Ming et al. 2007), and induces hyperexcitable states as seen in epilepsy, a comorbidity in approximately 20% of autistic cases (Bolton et al. 2011; Tuchman and Cuccaro 2011).
PCBs are OCs that had broad industrial uses, including use as adjuvants in paints and pesticide formulations (U.S. Environmental Protection Agency 2011). Although banned approximately 40 years ago, PCB exposures remain a concern to human health because of their persistence in the environment. Developmental and in vitro studies in rodents and nonhuman primates have demonstrated the ability of non-coplanar PCBs to cause imbalances in excitatory and inhibitory neurotransmission within critical regions for language development (Kenet et al. 2007), social cognition (Nakagami et al. 2011), and seizures (Kim and Pessah 2011; Kim et al. 2009). A substantial body of epidemiologic literature has provided evidence that cognitive deficits are associated with elevated PCB exposures, and more recently, elevated prenatal exposures to mono-ortho PCBs were found to be predictive of lower scores on both the Mental Development Index (MDI) and the Psychomotor Development Index (PDI) of the Bayley Scales of Infant Development (Park et al. 2010). Furthermore, an analysis of seven hydroxylated metabolites of PCBs in cord blood revealed that the metabolite from mono-ortho substituted PCBs were the only ones associated with reduced MDI and PDI scores (Park et al. 2009). These findings underscore the complexity of toxicities within a compound class and, by the same principle, the critical need to characterize differences among, for example, OPs or pyrethroids.
ACh-mediated neurotransmission is widely involved in the development of both the peripheral and the central nervous systems, and continues to play a critical role in regulating muscle movement, learning, attention, cognition, and memory throughout adulthood. ACh regulates aspects of nerve excitation and inhibition that influence brain plasticity, arousal, and reward. ACh increases excitation both directly and indirectly, and works through both nicotinic and muscarinic receptors to stimulate inhibitory interneurons, thereby modulating the activity of downstream effectors in a complex manner (Brown 2010; Scharf 2003).
Several cholinergic abnormalities have been reported in autism [Bauman and Kemper 2005; Perry et al. 2001; reviewed by Deutsch et al. (2010)]. In brief, studies of postmortem brain tissue have reported reduced nAChR binding in the frontal and parietal cortices (comparing 7 cases with 10 controls), reduced M1-muscarinic receptor binding in the parietal cortex (comparing 5 cases with 5 controls), and increased concentration of brain-derived neurotrophic factor (BDNF) (comparing 5 cases with 5 controls) (Deutsch et al. 2010). (BDNF is involved in the development and function of cholinergic neurons.) Although these studies involved small sample sizes, they suggest cholinergic abnormalities may be present in persons with autism.
OP insecticides irreversibly inhibit the active site of AChE, and while the severity of neurodevelopmental effects in animal studies correlate with AChE inhibition, additional neurotoxic effects have been observed at concentrations below the level sufficient to induce enzyme inhibition (Eddins et al. 2010; Levin et al. 2003; Slotkin et al. 2008). These effects include altered cell packing density, decreases in serotonin receptor and nAChR levels (Levin et al. 2010), altered Ca2+ and K+ ion concentrations (Harrison et al. 2002; Murgia 2004), and oxidative stress (Aluigi et al. 2005). Metabolism of OPs is mediated by the paraoxonase1 enzyme (PON1), whereby fast metabolizers suffer less AChE inhibition than slow metabolizers in response to the same level of exposure (Costa et al. 2005).
Pertinent to the male predominance observed in autism, sex selective developmental effects have been seen in animal models exposed to OPs. Chlorpyrifos exposure (1 mg/kg/day) in rats during postnatal days (PND) 1–4 decreased the number of errors in working and reference memory made by females, but increased the number of such errors made by males. These effects persisted into adolescence and adulthood, indicating a long-term consequence of exposure (Levin et al. 2001). Another study in rats showed that developmental exposures to low doses of the OP parathion induced greater developmental deficits in spatial navigation and working memory among males than females (Levin et al. 2009). Although these behaviors are not core features of autism, these findings provide evidence of different effects of early exposures between the sexes. In addition, parathion administration on PND1–4 at levels that barely inhibited cholinesterase was associated with deficits at 14–19 months of age, showing these deficits worsen with age (Levin et al. 2009).
The ability of OPs to inhibit AChE varies dramatically by chemical structure, which also determines reversibility. Aluigi et al. (2005) conducted a study examining the AChE-mediated developmental effects of OP exposure on chick embryos and discovered that 10–6 M chlorpyrifos was sufficient to inhibit head development. Even lower concentrations of chlorpyrifos-oxon disrupt axonal growth of rat dorsal root ganglia neurons (Yang et al. 2008), and sensory neuron development in zebrafish (Yang et al. 2011), indicating that exposure to very low levels of this OP has the potential to adversely influence development of neural networks (Yang et al. 2011). Persistent neurobehavioral consequences of chlorpyrifos exposure in zebrafish have also been demonstrated (Eddins et al. 2010; Levin et al. 2003). Although chlorpyrifos is still used worldwide in residential settings, residential use has been banned in the United States because of its neurotoxicity. However, no restrictions have been placed on its agricultural use.
Environ Health Perspect. 2012;120(7):944-951. © 2012 National Institute of Environmental Health Sciences