Novel dose-dependent alterations in excitatory GABA during embryonic development associated with lead (Pb) neurotoxicity

Abstract

Lead (Pb) is a heavy metal that is toxic to numerous physiological processes. Its use in industrial applications is widespread and results in an increased risk of human environmental exposure. The central nervous system (CNS) is most sensitive to Pb exposure during early development due to rapid cell proliferation and migration, axonal growth, and synaptogenesis. One of the key components of CNS development is the Gamma-aminobutyric acid (GABA)-ergic system. GABA is the primary inhibitory neurotransmitter in the adult brain. However, during development GABA acts as an excitatory neurotrophic factor which contributes to these cellular processes. Multiple studies report effects of Pb on GABA in the mature brain; however, little is known regarding the adverse effects of Pb exposure on the GABAergic system during embryonic development. To characterize the effects of Pb on the GABAergic system during development, zebrafish embryos were exposed to 10, 50, or 100 ppb Pb or a control treatment. Tissue up-take, gross morphological alterations, gene expression, and neurotransmitter levels were analyzed. Analysis revealed that alterations in gene expression throughout the GABAergic system and GABA levels were dose and developmental time point specific. These data provide a framework for further analysis of the effects of Pb on the GABAergic system during the excitatory phase and as GABA transitions to an inhibitory neurotransmitter during development.

Keywords: Developmental toxicity; GABA; Gene expression; Lead; Neurotoxicity; Zebrafish.

Fig. 1. Pb absorption in zebrafish tissue after embryonic exposure

Pb concentration in zebrafish tissue after exposure through 72 hpf to 0 (ND), 10 (295.61 ± 236.38 ng/g), 50 (611.52 ± 204.38 ng/g), or 100 (1139.49 ± 217.07 ng/g) ppb. Pb was undetectable in the control samples and significantly increased in a dose-response manner among the increasing Pb treatments (p<0.0001). (n=4; Different letters indicate that concentrations are significantly different from each other; Error bars depict standard deviation)

Fig. 2. GABA gene expression alterations following an embryonic Pb exposure

Results show the relative quantity of gene expression of all seven target genes at 24 (a), 48 (b), 60 (c), and 72 (d) hpf which were quantitatively compared between control embryos and those exposed to 10, 50, or 100 ppb Pb. Gene expression was normalized to gapdh. Pb exposure of 50 and 100 ppb caused the most significant alterations in gene expression at 24, 48, and 60 hpf. Minimal alterations occurred in gene expression at 72 hpf which agreed with our previous study (Peterson et al., 2011). (n=5-6; *p<0.05 **p<0.01; Error bars depict standard deviation)

Fig. 3. Alteration in GABA levels following embryonic Pb exposure

At 48 hpf (a), GABA levels were significantly different among the treatments (p<0.05). GABA levels in zebrafish in the control treatment were 78.31 ± 2.26 ng/fish and significantly increased in the 10 ppb treatment (88.43 ± 3.32 ng/fish). GABA levels in the 50 ppb treatment were 103.87 ± 8.18 ng/fish and were significantly increased compared to the control and the other Pb treatments. GABA levels in the 100 ppb treatment were not significantly different from the control treatment (83.14 ± 4.76 ng/fish). At 72 hpf (b), GABA levels in zebrafish in the control treatment were 79.17 ± 6.54 ng/fish. A significant decrease in GABA levels in all three Pb treatments was observed (p<0.05; 10 ppb: 74.86 ± 5.00 ng/fish, 50 ppb: 83.18 ± 8.17 ng/fish, and 100 ppb: 78.12 ± 4.69 ng/fish). (n=5; Different letters indicate Pb treatments that are significantly different from each other; Error bars depict standard deviation)

Fig. 4. Summary of GABAergic gene expression alterations following embryonic Pb exposure

Schematic representation of altered GABAergic gene expression at 10 (a), 50 (b), and 100 (c) ppb Pb at four developmental time points (24, 48, 60, and 72 hpf). Figures demonstrate the neuronal location and/or function of each gene throughout the GABAergic pathway. Arrows indicate direction of alterations in gene expression.GABA is represented by blue triangles