Developmental exposure to environmental levels of cadmium induces neurotoxicity and activates microglia in zebrafish larvae: From the perspectives of neurobehavior and neuroimaging

Highlights

• Zebrafish: a powerful model to visualize neurotoxicity of environmental pollutants.

• Cadmium exposure at environmental levels impairs zebrafish early development.

• Cadmium interferes with zebrafish locomotion and reactivity to environmental signals.

• Cadmium induces neurotoxicity and disrupts neurodevelopment of embryonic zebrafish.

• Cadmium activates zebrafish microglia and triggers neuroinflammation.

Abstract

Cadmium (Cd) is a worldwide environmental pollutant that postures serious threats to humans and ecosystems. Over the years, its adverse effects on the central nervous system (CNS) have been concerned, whereas the underlying cellular/molecular mechanisms remain unclear. In this study, taking advantages of zebrafish model in high-throughput imaging and behavioral tests, we have explored the potential developmental neurotoxicity of Cd at environmentally relevant levels, from the perspectives of neurobehavior and neuroimaging. Briefly, Cd²⁺ exposure resulted in a general impairment of zebrafish early development. Zebrafish neurobehavioral patterns including locomotion and reactivity to environmental signals were significantly perturbed upon Cd²⁺ exposure. Importantly, a combination of in vivo two-photon neuroimaging, flow cytometry and gene expression analyses revealed notable neurodevelopmental disorders as well as neuroimmune responses induced by Cd²⁺ exposure. Both cell-cycle arrest and apoptosis contributed jointly to a significant decrease of neuronal density in zebrafish larvae exposed to Cd²⁺. The dramatic morphological alterations of microglia from multi-branched to amoeboid, the microgliosis, as well as the modulation of gene expression profiles demonstrated a strong activation of microglia and neuroinflammation triggered by environmental levels of Cd²⁺. Together, our study points to the developmental toxicity of Cd in inducing CNS impairment and neuroinflammation thereby providing visualized etiological evidence of this heavy metal induced neurodevelopmental disorders. It's tempting to speculate that this research model might represent a promising tool not only for understanding the molecular mechanisms of Cd-induced neurotoxicity, but also for developing pharmacotherapies to mitigate the neurological damage resulting from exposure to Cd, and other neurotoxicants.

Introduction

Cadmium (Cd) as a biologically non-essential heavy metal is discharged into ecosystems via various pathways including metal refining, fuel burning, as well as utilization of plastic stabilizers, phosphors, pesticides and paints, thereby is widely distributed in air, soil and water. For example, the dissolved Cd levels measured around the world ranged from 1 to 100 μg/L, while in seriously polluted rivers ranged from 60 to 300 μg/L (Campoy-Diaz et al., 2020; Singh and Chandra, 2019; Tripathy et al., 2022; Wright and Welbourn, 1994). With the process of industrialization, increasing amount of Cd is absorbed thus accumulated in living organisms (Larsson and Wolk, 2016). Recently, growing evidence has shown that Cd accumulation increases the risk of neurological disorders. For instance, it has been shown to damage nervous tissue and cells (Jiang et al., 2015; Chow et al., 2008; Song et al., 2021) thus representing one of the risk factors of Parkinson's and Alzheimer's diseases (Li et al., 2017; Notarachille et al., 2014); in addition, increased Cd levels in the blood may not only enhance the incidence of ischemic stroke in adults (Wen et al., 2019), but also are significantly associated with an increased risk of attention-deficit/hyperactivity disorder (ADHD) in their offspring (Kim et al., 2020). In short, Cd has been widely acknowledged to pose great threats to the central nervous system (CNS), however, the underlying cellular and molecular mechanisms remain to be further elucidated. In this study, we explored deeply the neurotoxicological mechanisms of Cd by using zebrafish as the model system.

Zebrafish is a vertebrate model organism widely used in genetic, developmental and neurological studies because of its distinct advantages like rapid development, optical transparency of embryos, and the sufficient homology with human at both molecular and genomic levels (Kalueff et al., 2014; Wragg and Müller, 2016). Importantly, various transgenic lines expressing fluorescent proteins provide powerful tools for non-invasive imaging and tracking of numerous cell types (Kurita et al., 2004; Zon and Peterson, 2005). Therefore, zebrafish represents an ideal model for assessing the effects of environmental toxins on the developing organisms. It has been recently used in Cd toxicologic studies to define developmental toxicity as well as the underlying mechanisms, for instance, Cd exposure affected normal development of zebrafish larvae (Liu et al., 2016; T. Zhang et al., 2021), damaged the olfactory sensory neurons (Heffern et al., 2018), induced degeneration of neurons and astrocytes in the brain (Monaco et al., 2016, 2017; Song et al., 2021). However, few studies have investigated comprehensively the Cd-modulated CNS from the perspective of neurobehavior and neuroimaging. Particularly, previous studies have focused more on neurons, whereas very little is known about Cd effects on microglia, the important resident immune cells continuously monitoring the CNS homeostasis. Therefore, this study aimed at visualizing the effects exerted by Cd on two main types of cells, neurons and microglia present in the zebrafish brain, so as to shed light on the causes of neurotoxicity induced by this worldwide existed pollutant.

In this work, we exposed zebrafish embryos to environmentally relevant levels of Cd, and then investigated embryonic developmental toxicity, larval neurobehavior, morphology and structure of neurons and microglia, as well as the related gene expression profiles. Our results revealed multiple connections between Cd exposure and neurodevelopmental disorders as well as neurotoxic reactions thus providing novel evidences of the Cd toxicity from a fresh perspective of neurobehavior and neuroimaging. Zebrafish model with distinct advantages in vivo imaging and high-throughput behavioral tests, together with this research paradigm represent promising tools not only for understanding the mechanisms of environmental pollutants induced neurotoxicity, but also for developing pharmacotherapies to mitigate the neurological damages resulting from exposure to various neurotoxicants.