Opportunities and challenges for using the zebrafish to study neuronal connectivity as an endpoint of developmental neurotoxicity

Abstract

Chemical exposures have been implicated as environmental risk factors that interact with genetic susceptibilities to influence individual risk for complex neurodevelopmental disorders, including autism spectrum disorder, schizophrenia, attention deficit hyperactivity disorder and intellectual disabilities. Altered patterns of neuronal connectivity represent a convergent mechanism of pathogenesis for these and other neurodevelopmental disorders, and growing evidence suggests that chemicals can interfere with specific signaling pathways that regulate the development of neuronal connections. There is, therefore, a growing interest in developing screening platforms to identify chemicals that alter neuronal connectivity. Cell-cell, cell-matrix interactions and systemic influences are known to be important in defining neuronal connectivity in the developing brain, thus, a systems-based model offers significant advantages over cell-based models for screening chemicals for effects on neuronal connectivity. The embryonic zebrafish represents a vertebrate model amenable to higher throughput chemical screening that has proven useful in characterizing conserved mechanisms of neurodevelopment. Moreover, the zebrafish is readily amenable to gene editing to integrate genetic susceptibilities. Although use of the zebrafish model in toxicity testing has increased in recent years, the diverse tools available for imaging structural differences in the developing zebrafish brain have not been widely applied to studies of the influence of gene by environment interactions on neuronal connectivity in the developing zebrafish brain. Here, we discuss tools available for imaging of neuronal connectivity in the developing zebrafish, review what has been published in this regard, and suggest a path forward for applying this information to developmental neurotoxicity testing.

Developmental exposure to silver nanoparticles at environmentally relevant concentrations alters swimming behavior in zebrafish (Danio rerio)

“…Zebrafish models have also been used to show that Ag-NPs accumulate in brain tissue (Asharani 2008) and alter the expression of genes related to central nervous system (CNS) development (Xin 2015). Because of their ability to penetrate the chorion (Lee 2007, Lee 2013), Ag-NPs pose a significant threat to early-stage zebrafish embryos when many neurodevelopmental processes are at their peak (Miller 2018). …”

The presynaptic glycine transporter GlyT2 is regulated by the Hedgehog pathway in vitro and in vivo

“…Zebrafish express homologs for more than 70% of human genes and their molecular underpinnings controlling neurotransmission are conserved with higher vertebrates 98 . For example, properties of glycinergic biology in zebrafish mimic well the functions observed in mammals, as for example 1) GlyT1 zebrafish mutants present excessive glycinergic transmission due to the lack of glycine recapture 74,99 , 2) loss of glycine receptor function results in an hyperekplexia-like phenotypes 100,101 and 3) GlyT2 can be used as a reliable marker for glycinergic neurons 40,41 .…”

Generation of a Triple-Transgenic Zebrafish Line for Assessment of Developmental Neurotoxicity during Neuronal Differentiation

“…However, while rodents have contributed significantly to our understanding of DNT [3,[17][18][19], experiments using large numbers of rodents are time-consuming, expensive, and accompanied by ethical concerns. Thus, alternative test systems, such as human stem/progenitor cell and zebrafish models, have the potential to enable testing of large chemical libraries while reducing expense and minimizing rodent use [4,[20][21][22][23][24][25].In human stem/progenitor cell models, various endpoints have been used to assess DNT, including viability, apoptosis, proliferation, migration, differentiation, and neuronal network formation [24,26,27]. These endpoints facilitate the identification of adverse outcome pathways and the development of integrated approaches to test and assess chemical-induced DNT [22,28].…”

“…However, distinguishing between multiple cell types requires a multiplexed fluorescent reporter system and, although this has been employed in some human stem/progenitor cell models [31], to our knowledge, it has not been used to distinguish between neurons, astrocytes, and oligodendrocytes in the same organism.Compared with other species, zebrafish offer several advantages as a model system for DNT testing. For example, the pattern of developmental gene expression and structure of various brain regions is relatively conserved in zebrafish; a wide range of chemicals can be absorbed from the surrounding medium; the small size and prolific breeding capacity lowers housing and experimental costs [4,23,[32][33][34][35]; and the optical transparency, particularly of pigmentless mutants, make zebrafish suitable for in vivo fluorescence imaging [4,20]. To date, many transgenic (Tg) zebrafish lines have been developed that express fluorescent proteins in specific neuronal subtypes [36][37][38], and multiplexed reporter systems have also been implemented in this species [39].…”

“…However, while rodents have contributed significantly to our understanding of DNT [3,[17][18][19], experiments using large numbers of rodents are time-consuming, expensive, and accompanied by ethical concerns. Thus, alternative test systems, such as human stem/progenitor cell and zebrafish models, have the potential to enable testing of large chemical libraries while reducing expense and minimizing rodent use [4,[20][21][22][23][24][25].…”