Early findings suggested that chronic Pb²⁺ exposure could produce its behavioral effects by altering synaptic development. Mammalian studies reported a variety of alterations in synaptic morphology and physiology in the brains of animals exposed to Pb²⁺ during development (Petit and LeBoutillier, 1979; Kiraly and Jones, 1982; Altmann et al., 1993). Today, the synapse remains a focus for studying the effects of toxins on brain development. Here the Drosophila larval neuromuscular junction (NMJ) offers a distinct advantage since one can compare the same, identified synapse in controls and treated animals (Figure 2). Note that this neuromuscular system is unique even amongst invertebrates because there is a stereotypic pattern of neuromuscular connections where both the presynaptic (motor neuron) and postsynaptic cells (muscle fiber) can be uniquely identified (Keshishian et al., 1996). These neuromuscular synapses have been used to demonstrate the effects of second messengers, cell-adhesion molecules and their modulators, and impulse activity on synaptic development (Budnik, 1996; Budnik et al., 1996; Davis et al., 1996).

We found that indeed one could detect synaptic abnormalities resulting from chronic Pb²⁺ exposure at the Drosophila larval NMJ. Initial studies found that the motor terminal formed by motoneuron RP3 on Muscle Fiber 6 (MF6) showed greater variability in size for animals raised in media containing Pb²⁺ compared to controls (Morley et al., 2003). One could imagine that increasing the variability of synaptic size could result in a loss of fine-tuning of synaptic function; this effect, if seen in the brain, would likely be detrimental to circuit dynamics and behavior. It is noteworthy that this synaptic change was subtle and it likely would have been difficult to detect when making comparisons among a large population of synapses in the mammalian brain. Given that the behavioral effects of toxins, such as Pb²⁺, can be subtle, it seems reasonable that the effects on synapses might also be small.

Pb²⁺ affects proteins that bind Ca²⁺ including those that regulate the intracellular Ca²⁺ concentration ([Ca²⁺]_i), such as Ca²⁺ channels and Ca²⁺ pumps. Changes in Ca²⁺ regulation could be particularly important for the synapse since [Ca²⁺]_i controls multiple steps in synaptic development; e.g., growth cone guidance (Jin et al., 2005), synapse formation (Xu et al., 2009), and synapse elimination and stabilization (Pratt et al., 2003; Lohmann and Bonhoeffer, 2008). In addition, altered Ca²⁺ regulation could influence transmitter release (Zucker, 1996) and both long-term and short-term forms of synaptic plasticity at the mature synapse (Zucker and Regehr, 2002; MacDonald et al., 2006).

Acute Pb²⁺ exposure blocks Ca²⁺ channels. This has been demonstrated for a variety of voltage-dependent Ca²⁺ channels in both invertebrates (Audesirk and Audesirk, 1989) and mammals (Evans et al., 1991). The acute application of micromolar concentrations of Pb²⁺ can reduce the activity of the plasma membrane Ca²⁺ ATPase (PMCA) in humans and rats (Mas-Oliva, 1989; Sandhir and Gill, 1994a, b); however the PMCA can be stimulated by low Pb²⁺ concentrations (Mas-Oliva, 1989; Campagna et al., 2000). In addition, chronic in vivo Pb²⁺ exposure has been found to produce a persistent inhibition of PMCA activity in human erythrocytes (Campagna et al., 2000) and rat synaptosomes (Sandhir and Gill, 1994a, b).

We studied the effect of chronic Pb²⁺ exposure on presynaptic Ca²⁺ regulation at the RP3-to-MF6 synapse in Drosophila larvae (He et al., 2009). Ca²⁺ indicators were loaded in these motor terminals and we measured the changes in [Ca²⁺]_i produced by single action potentials and action potential trains (Figure 2). We found that chronic exposure to Pb²⁺ resulted in a greater increase in [Ca²⁺]_i during trains of action potentials and a slower decay of postsynaptic [Ca²⁺]_i at the end of the train. This is likely due to a decrease in the activity of the PMCA and it provides an interesting parallel to the effect of Pb²⁺ exposure on the PMCA in mammals. The direct effect of this large increase in [Ca]_i was that the Pb²⁺-exposed animals showed greater synaptic facilitation. This was consistent with the residual Ca²⁺ model for synaptic facilitation (Zucker and Regehr, 2002) and with mammalian studies showing that knocking down expression of the PMCA resulted in enhanced synaptic facilitation (Empson et al., 2007). In that study, reduced PMCA expression also produced changes in neuronal structure (Empson et al., 2007) and it may be that the Pb²⁺-induced changes in the structure of the larval NMJ resulted from reduced PMCA activity and altered Ca²⁺ regulation.

In summary, identified NMJs in Drosophila larvae allow for the detection of subtle changes in synaptic structure and function resulting from developmental exposure to toxins. Our evidence suggests that chronic Pb²⁺ exposure produces parallel synaptic changes in Drosophila and mammals; this would not be surprising given that chemical synapses found in vertebrates and invertebrates are very similar.

Developmental neurotoxicology research requires an approach that reduces the candidate toxin-regulated genes to a manageable number. Fortunately, a new multi-dimensional strategy has been developed, called genetical genomics, which identifies master modulatory loci that regulate the expression of hundreds of other genes (Jansen and Nap, 2001; Broman, 2005; de Koning and Haley, 2005; Li and Burmeister, 2005; Rockman and Kruglyak, 2006). Genetical genomics combines two methodologies, microarray-based whole transcriptome analyses and extracting QTLs by using RILs. Global gene expression levels are determined for each RIL, and then QTL mapping software (e.g., R/QTL; Broman et al., 2003) is used to find above-threshold statistical associations between specific chromosomal loci and transcript levels for all the genes measured on the microarrays. With QTL analysis, we can identify the specific chromosomal loci that regulate genes, which are referred to as eQTLs (Mueller et al., 2006; West et al., 2007; Majewski and Pastinen, 2011; Zhang et al., 2012).

Our research is the first that combines genetical genomics with toxicogenomics, an experimental approach we call “genetical toxicogenomics” (Ruden et al., 2009). Li et al. (2008) showed that adding environmental perturbations in genetical genomics studies, as we did in our study (Ruden et al., 2009), allows toxin-response genes to be identified. We believe that this genetical toxicogenomics approach will drive the field of toxicology and aid in understanding the effects of toxins such as lead.

Expression QTL analyses were performed for all ~18,000 genes and other microarray features by treating the expression level of each gene as a quantitative trait. The genetics of gene expression in RILs can be mapped as eQTLs. Flies from each of 75 RILs were fed, from egg to adult, either control food or lead-treated food (made with 250 μM lead acetate). RNA expression analyses of whole adult male flies (5–10 days old) were performed with Affymetrix Dros2 whole genome arrays (18,952 probe sets). Loci that are linked to a gene are called locally acting or cis-eQTL, whereas loci that are distantly acting are called trans-eQTLs (Broman, 2005).

The first genetical genomics study to identify genes with significant GxE interactions was done in Caenorhabditis elegans; the authors identified a group of genes with trans-eQTL that are induced by heat shock, which they called plastic QTL (Li et al., 2006). Smith and Kruglyak (2008) recently performed a detailed analysis of GxE-eQTL in yeast (which they call “gxeQTL”) grown in either glucose or ethanol as the sole carbon source. Others have identified GxE interactions in which the environment is a different tissue (e.g., brain vs. liver; Hovatta et al., 2007). In our studies, among the 1,389 genes with cis-eQTL, there were 405 genes unique to control flies and 544 genes unique to lead-treated ones (440 genes had the same cis-eQTLs in both samples).

There were 2,396 genes with trans-eQTL that mapped to 12 major hotspots that met statistical significance (p < 0.05, chi-squared test, based on permutation analyses, on 5-cM windows). Unexpectedly, we identified two hotspots, one located on the second chromosome (polytene region 30AB) and one on the third chromosome (polytene region 73D), which co-regulate 33 genes, all of which are induced by lead (Figure 3). We have shown by QTL analysis that marker locus 30AB contributes to lead-dependent changes in locomotion, which suggests that the genes in the 30AB hotspot can be used as a functional test to identify both the lead-dependent trans-regulatory factor and the common cis-regulatory motifs (Hirsch et al., 2009). We propose that a trans-regulator located at 73D increases expression of its target genes when it binds lead, regardless of the genotype of the 73D hotspot. In contrast, a second trans-regulator located at 30AB increases expression of the co-regulated target genes only when it has the ORE genotype (Figure 3).

This model can explain the data, but other explanations are possible. For example, the putative trans-regulator could increase the stability of the mRNA of the target genes, or there could be an indirect effect on steady-state mRNA levels. MEME (Multiple Em for Motif Elicitation) analyses of the genes regulated by the 30AB and 73D trans-regulators identified both conserved proximal promoter and 3′-untranslated region (UTR) sequences (data not shown). Fine-mapping of the genes that underlie the QTLs and molecular and biochemical analyses should enable us to determine the mechanism involved.

In this review, we describe recent results from several laboratories that are collaborating on studying the effects of lead on the developing Drosophila nervous system. The Possidente and Hirsch laboratories study the effects of lead on behavioral quantitative traits. The Lnenicka laboratory studies the effects of lead on the NMJ in the larvae. The Ruden, Lu, and Garfinkel laboratories study the effects of lead on gene expression using quantitative genetics techniques.

Further innovations to the field of genetical genomics will make use of next-generation RNA sequencing (RNA-seq) rather than gene expression microarrays. RNA-seq will allow us not only to accurately determine gene expression levels, but also to analyze alternative splicing products of genes, thereby adding a new dimension to these studies. Since over 90% of human genes and a similar percentage of Drosophila genes are alternatively spliced, RNA-seq could provide some exciting and novel findings about environmental regulation of alternative mRNA splicing. The potential for RNA-seq to supplant microarrays requires, however, the development of robust statistical tools for analyzing the resulting huge datasets.

Adding metabolomics profiling to these studies will allow us to identify metabolite QTL (mQTL) that are specific for lead. To our knowledge mQTL analyses have never been conducted in any organism, although genome-wide association studies combined with metabolic profiling were conducted in a recent human study (Prakash, 2011; Suhre et al., 2011). The human study identified 37 genetic loci associated with blood metabolite concentrations, of which 25 showed effect sizes of 10–60% which is extraordinarily high for genome-wide association studies (Prakash, 2011; Suhre et al., 2011).

We are now entering the stage of understanding the complex genetic pathways that are affected by developmental lead exposure. The sophisticated genetic analyses that are possible in Drosophila will soon allow us to manipulate these pathways to better understand how they are affected by lead. The sequencing of several Drosophila strains and studying the effects of lead on neurodevelopment in these strains will allow us to better understand the evolution of lead-sensitive pathways. Together, these studies will provide a better understanding of the health effects of lead in humans.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.