THE NEURODEVELOPMENTAL TOXICITY OF HEAVY METALS: A FISH PERSPECTIVE

Abstract

The causes of neurodegenerative diseases are complex with likely contributions from genetic susceptibility, and environmental exposures over an organism’s lifetime. In this review, we examine the role that aquatic models, especially zebrafish, have played in the elucidation of mechanisms of heavy metal toxicity and nervous system function over the last half-decade. Focus is applied to cadmium, lead and mercury as significant contributors to central nervous system morbidity, and to the application of numerous transgenic zebrafish expressing fluorescent reporters within specific neuronal populations or brain regions enabling high-resolution neurodevelopmental and neurotoxicology research.

INTRODUCTION

The vertebrate central nervous system (CNS) is an evolutionarily conserved system consisting of the brain and spinal cord. During embryogenesis, the brain of all vertebrates partitions along the anterior-posterior axis into the forebrain (prosencephalon), consisting of the telencephalon and diencephalon; midbrain (mesencephalon); and hindbrain (rhombencephalon), consisting of the metencephalon and myelencephalon, which then transition to the spinal cord. In mammals, the telencephalon expands substantially and envelops both the diencephalon and mesencephalon and becomes the cerebral cortex or cerebrum, where the seat of consciousness appears to reside¹, and where voluntary movement is controlled, and learning, memory, language, and sensory processing occur. In other vertebrates including fish, the telencephalon is a considerably smaller structure situated anterior to the mesencephalon and from which the more prominent olfactory bulb projects. In addition to this major difference, the adult brain of fish, reptiles, amphibians and birds differs anatomically from the mammalian brain in that the three major subdivisions of the brain (fore-, mid- and hindbrain) remain situated along the anterior-posterior axis of the vertebrate body in contrast to the folding of the fore- and mid-brain into a single, complex structure in mammals, thus exhibiting a simplified architecture relative to their mammalian counterparts. Neurodevelopmental disorders (NDDs) can be broadly defined as defects in growth or development of the central nervous system, which can be caused by genetic or environmental factors. The latter can include physical trauma, exposure to xenobiotics, and biological causes such as viral or bacterial infections² during critical periods of nervous system development. In humans, manifestations of neurodevelopmental disorders are wide-ranging and complex, and include intellectual disabilities, communication disorders, traumatic brain injuries, and autism spectrum disorders, epilepsies, and motor and coordination disorders. Many of these human disorders appear to have model organism counterparts including rodents and fish, thus enabling experimentation designed to elucidate the mechanistic bases of their origins. Although outside of the scope of this review, the reader is referred to several excellent reviews discussing the application of model organisms towards understanding complex human neurodevelopmental disorders^3–6.

In this review, we focus on describing transgenic zebrafish generated over the last decade, in which specific neuronal populations are labeled with fluorescent tags for in vivo visualization of normal and pathological neurodevelopmental processes, and we review the effect of cadmium (Cd), lead (Pb) and mercury (Hg), on neurodevelopment and neurodevelopmental outcomes by specifically focusing on the contributions that aquatic species, mainly fish, have made toward our understanding of the role these metals have on adverse neurological outcomes in affected populations.

TRANSGENIC ZEBRAFISH USED IN THE STUDY OF NEURODEVELOPMENT

Transgenic zebrafish in which specific neuronal populations or CNS regions are labeled with fluorescent reporters have provided important insights into neurodevelopment, and are a promising resource for understanding the effects of neurotoxic compounds on brain function. The transgenic lines discussed below are summarized in Table 1.

Table 1.

List of Transgenic Zebrafish Lines

Gene Symbol	Genotype	Reporter	Type	Neurological Endpoint	Reference
mt	Tg(UAS:mtPAGFP:mtDsRed2)	GFP/dsRed2	Structural	Dopaminergic axons and mitochondrial transport	Dukes et al. (2016)
gfap	Tg(gfap:GFP)mi2001	GFP	Structural	Adult neural stem cell behavior and Müller glia	Bernardos and Raymond (2008)
th2	Tg(th2:GFP-dlx5/6:mCherry)	GFP/mCherry	Structural	Dopaminergic neurons from embryonic neural precursors	McPherson et al. (2016)
slc18a2	Tg(ETvmat2:GFP)	GFP	Structural	Monoaminergic neurons	Wen et al. (2015)
mpz	Tg(mpz:EGFP)	GFP	Structural	CNS oligodendrocytes	Bai et al. (2014)
gap43	Tg(GAP43:GFP)	GFP	Structural	Optic nerves	Udvadia et al. (2008)
hsp70	Tg(hsp70:GFP)	GFP	Structural	Olfactory neurons	Halloran et al. (2000)
olig2	Tg(olig2:EGFP)	GFP	Structural	Oligodendrocytes	Shin et al. (2003)
pomca	Tg(-1.0pomca:GFP)	GFP	Structural	Corticotropic cells	De Marco et al. (2016)
kctd12.2	Tg(UAS:kctd12.2:mt)vu442	GFP	Structural	Habenular nuclei	Taylor et al. (2011)
ascl1a	Tg(ascl1a:GFP)	GFP	Structural	Müller glia and retinal regeneration	Wan et al. (2012)
th2	Tg(th2:Gal-VP16-UAS-E1b:NTR-mCherry)	Gal/mCherry	Structural	Hypothalamic neurons	McPherson et al. (2016)
arxa	Tg(arxa:mCherry-ARX_enhancer:Kal4)	mCherry	Structural	Forebrain	Ishibashi et al. (2015)
tau	A152T-tau	none	Neural Degeneration	Neurodegeneration and proteasome compromise	Lopez et al. (2017)
C9orf72	C9orf72 associated repeat	GFP	Neural Degeneration	Dipeptide repeat protein associated toxicity in ALS/FTLD	Ohki et al. (2017)
ca8	Tg(ca8:FMA-TagRFP-2A-casp8ERT2)	RFP	Neural Degeneration	Target ablation of cerebellar Purkinje cells	Weber et al. (2016)
tau	Tg(tau-GFP)	GFP	Neural Degeneration	Neurodegeneration by tau proteins	Wu et al. (2016)
ctnna	ct3aGt	Citrine (YFP)	Functional	Cadherin-mediated based hindbrain cell-cell interactions	Žigman et al. (2011)
GCaMP5G	GCaMP5G calcium indicator	optoacoustic	Functional	Neural activity	Deán-Ben et al. (2017)
fhf1b	mutant FHF1B	gain of function	Functional	Early-onset epileptic encephalopathies	Siekierska et al. (2016)
fezf2	Tg(fezf2-GFP)	GFP	Functional	Neural stem cells proliferation	Berberoglu et al. (2009)

Transgenics that Label Specific Neurons

The ability to generate stable transgenic zebrafish that label specific neuronal populations or particular regions of the brain has been an extremely useful tool to study neurodevelopment in the presence of toxins and toxicants by tracking neuronal outgrowth and circuit formation, and by quantifying changes in fluorescence during exposure as evidence of abnormal neuronal function^7,8. Examples include double-labeling mitochondria to measure mitochondrial transport, fusion and fission in dopaminergic neuronal axons⁹; visualizing cadherin bases cell-cell interactions in the hindbrain with the ctnna promoter¹⁰; tagging specific neurons with stable GFP expression, including dopaminergic neurons with the th2 promoter¹¹, monoaminergic neurons with the slc18a2 promoter¹², and habenular nuclei with the kctd12.2 promoter to assess monoamine regulation and study asymmetric brain development^13,14; and, neural stem cell proliferation and response to neural injury with the fezf2 promoter¹⁵. Another increasingly important area of research relates to sensory neurons and their role in behavior, learning, and emotional disorders^16–18. Multiple transgenic zebrafish have been developed to study a variety of sensory systems by fluorescently labeling optic nerves using the gap43 promoter¹⁹; olfactory neurons using the inducible hsp70 promoter²⁰; and single-cell resolution studies of adult telencephalic neural stem cells using the gfap promoter^21,22. Recently, a transgenic line in which GFP is expressed under the control of enhancers of the arxa gene was used to demonstrate that diminished dosage of the arxa gene transcript affects neuronal outgrowth and path finding capabilities resulting in neurodevelopmental disturbances similar to those observed in patients with copy number variations of the human ortholog, ARX²³; therefore, opening up possibilities for using this transgenic to characterize xenobiotics that could impact gene dosage and lead to new therapies. Finally, the impact of support cells, including glial cells, oligodendrocytes, and corticotropic cells should not be underestimated as these cells outnumber neurons 10:1²⁴. Transgenic lines for support cell imaging include mpz, and olig2, which label oligodendrocytes^25–28; pomca, which labels corticotropic cells in the anterior pituitary^29–31; and gfap, which allows visualization of radial-glial cells³².

Transgenics that Alter Neuronal Function

While the transgenic lines listed above can be used to visualize cell types, and investigate chemical influence, it is also possible to assess how gain or loss of function mutations impact toxicant susceptibility. An evaluation of viral insertional mutants and their related phenotypes uncovered novel genomic lesions resulting in defects in gliogenesis, glial patterning, neurogenesis, and axon guidance that may be useful in future studies of neuronal function under different environmental conditions³³. Other gain or loss of function transgenics include the loss of function mutant hdac1^hi1618, which regulates neural progenitor differentiation into neurons, and glial-dependent myelination through integration of the Hedgehog, Notch, and Wnt signaling pathways^34–35; a genetically-encoded calcium indicator, GCaMP5G, that coupled with optoacoustic imaging enables visualization of calcium-based neuronal activity³⁶; and a gain of function mutant of fhf1b, which causes early-onset epileptic encephalopathy, and may be a useful mutant for studying chemical influence on voltage-gated sodium channels³⁷.

Transgenics to Study Neural Degeneration

Advances in public health have led to greater longevity giving rise to an increased number of people suffering from neurodegenerative diseases, including Parkinson’s and Alzheimer’s disease³⁸. Although robust causal factors for these increases have eluded epidemiological analyses, the accumulation of exposures over prolonged lifespans is one possible mechanism suspected for the observed increase in neurodegenerative diseases³⁹. Zebrafish have emerged as an important tool to study neurodegenerative diseases⁴⁰, resulting in the development of new transgenic lines that may be used to study these diseases. For example, mutations in MAPT, which encodes the tau protein, have been causally associated with frontotemporal dementia⁴¹. A commonly occurring human tau mutation, A152T-tau, consisting of a single G > A nucleotide change, diminishes tau binding to microtubules and increases neurofibrillary tangle formation. Recently, this mutation has been introduced into zebrafish and observed to cause neurodegeneration and proteasomal deficiencies, which could be partially rescued by pharmacologically upregulating autophagy⁴¹. A second tau transgenic has been used to study factors influencing oligomer formation and disease progression⁴². A second devastating neurological disorder, amyotrophic lateral sclerosis (ALS), is often associated with an expansion of a GGGGCC repeat in a non-coding region of the chromosome 9 open reading frame 72 (C9orf72) locus⁴³. Recently, a zebrafish C9orf72-GFP transgenic was developed as a means to characterize therapeutic interventions for ALS⁴³, and could potentially be used to better understand the influence of toxic insults on disease onset and progression. Many neurodegenerative diseases are marked by programmed cell death, and having an effective and efficient way to replicate the disease progression could be instrumental in developing treatments⁴⁴. This line of research could benefit from established transgenic zebrafish such as a recently developed line that allows for cell type-specific caspase-mediated (ca8) apoptosis based on a tamoxifen-inducible system⁴⁴.

OVERVIEW OF HEAVY METALS AND NEUROTOXICITY

Heavy metals are naturally occurring metals exhibiting high atomic weights and high densities. Many heavy metals, including copper, iron, manganese, nickel, selenium, and zinc have important biological roles as cofactors for numerous proteins and enzymes. However, a significant number of heavy metals have no known biological roles, including cadmium, lead, and mercury but instead exhibit highly toxic properties when consumed by animals, including humans, and are classified as toxic heavy metals. Although naturally occurring, human activities, particularly through industrial processes, have led to widespread distribution of toxic metals throughout the biosphere. The widened distribution range increases the likelihood that humans will be exposed to toxic heavy metals through air, water, contaminated soil, and food. The World Health Organization lists cadmium, lead, and mercury in its list of top 10 chemicals of major public health concern⁴⁵, and exposure to these metals has been linked to numerous neurodevelopmental and neurodegenerative disorders in humans^46,47. In the following section, we summarize the latest research on the neurotoxicity of these three elements, with emphasis on the contributions made by aquatic models, primarily zebrafish, to our understanding of their toxic mechanisms.

PATHOLOGICAL EFFECTS OF HEAVY METALS ON FISH NEURODEVELOPMENT

Cadmium (Cd)

A variety of fish species have been used to study the effects of Cd exposure, including fathead minnow, rainbow trout, and sea bass^48–51. These studies, summarized in Table 2, were performed in larvae, juveniles and adults, and showed that Cd is capable of increasing auditory thresholds, increasing growth rates, impairing social and escape behavior, accumulating in the olfactory bulbs, and damaging the sensory macula and neuromast^48–51. Even at very low levels (1.9 ppb), Cd accumulates in the brain, causing an increase in expression of apoptotic genes (e.g., c-jun), and detoxifying genes (e.g., mt1 and mt2)⁵². This trend continues as the dose is increased to 200 ppb, as long as the exposure time remains less than 24 hours with one study showing an induction of the antioxidant gene, nrf-2, in the olfactory bulb and telencephalon⁵³ and another showing induction of mt2 and smtb in the brain⁵⁴. As the exposure time (2 – 30 days) and concentration increase (180 – 1000 ppb), these protective mechanisms appear to be overwhelmed and signs of stress and tissue damage appear^55–58. This damage includes changes in retinal neuronal morphology, increased sensitivity to light, decreases in glial fibrillary acidic protein (an astroglial cell cytoskeleton protein), and increases in reactive oxygen species (ROS), nitric oxide, and malondialdehyde^55–58. The LC50 levels for Cd are approximately 27 ppm, which is coupled to AChE inhibition⁵⁹, whereas brain homogenates exposed to LC50 levels for 10 minutes show evidence of nucleotide hydrolysis⁶⁰, indicative of DNA damage. In larval zebrafish the trend seems similar, including increases in mt2 and smtb expression⁶¹ followed by oxidative stress, abnormal histology, immunotoxicity, cell death, and a reduction in olfactory-dependent predator responses seen at mid-range levels (112 – 970 ppb)^20,62,63. These data suggest that in adult and larval fish, Cd exposure induces oxidative stress that at high doses or chronic exposures overwhelms natural defense systems leading to systemic damage, possibly through apoptotic mechanisms.

Table 2.

Summary of Studies Measuring Neurological Effects of Cadmium (Cd) Exposure in Fish

Exposure Duration	Age at Exposure	Species	Chemical	LOEC1(ppb2)	Major Endpoints	Reference
4d	Adult	Fathead minnows	Cd3	2.5	Increase in auditory threshold	Low and Higgs (2015)
1d	2–5 dpf	Rainbow trout	Cd3	2	Higher growth rate, altered social behavior & olfactory accumulation	Sloman et al. (2003)
4h/day for 8d	Juveniles	Sea bass	Cd3	0.5	Sensory macula damage and impaired escape behavior	Faucher et al. (2008)
4h	Juveniles	Sea bass	Cd3	5	Neuromast damage	Faucher et al. (2006)
21d	Adults (Males)	Zebrafish	Cd3	1.9	Increased apoptotic (c-jun) & detoxifying genes (mt1 & mt2) at 21d	Gonzalez et al. (2006)
1d	Adults	Zebrafish	Cd3	110	Induction of nrf2 antioxidant genes (increased olfactory neuron cell death)	Wang and Gallagher (2013)
30d	Adults	Zebrafish	Cd chloride(CdCl2)	183	Changes in retinal morphology & ultrastructure, increased light sensitivity	Avallone et al. (2015)
1 – 6h	Adults	Zebrafish	Cd3	200	Increased mt2 and smtb	Wu et al. (2016)
2d, 7d, 16d	Adults	Zebrafish	CdCl2	613	Decrease in glial fibrillary acidic protein	Monaco et al. (2016)
1d, 4d	Adult (Females)	Zebrafish	Cd3	1000	Increased ROS, nitric oxide, & malondialdehyde in brain and liver	Zheng et al. (2016)
3d	Adult (Females)	Zebrafish	Cd3	1000	Induction of mt2, smtb, and accumulation of Cd in ovaries and F1 larvae at 72hpf	Wu et al. (2012)
2d	Adults	Zebrafish	CdCl2	26122	LC50 and AChE inhibition	Zhang et al. (2017)
10m	Adult Brain	Zebrafish	Cd acetate Cd(CH3COO)2	28102	Nucleotide hydrolysis	Senger et al. (2006)
2d	3–5 dpf	Zebrafish	CdCl2	9	Increased mt2 and smtb	Wu et al. (2008)
4d	3–7 dpf	Zebrafish	Cd3	112	Cell death, altered histological and changes in olfactory dependent behavior.	Matz and Krone (2007)
3h	80 – 83 hpf	Zebrafish	Cd3	562	Reduced olfactory dependent predator response	Blechinger et al. (2007)
12h	Juvenile	Zebrafish	Cd3	970	Increased ROS and immunotoxicity	Zheng et al. (2017)
50d	0–50 dpf	Zebrafish	Cd3	20	Lower survival at 24 hpf and reduced olfactory-dependent predator response (64 dpf)	Kusch et al. (2008)
3d	0–72 hpf	Zebrafish	CdCl2	112	cyp19a1b gene expression and anti-estrogenic activity	Chouchene et al. (2016)
3d	0–72 hpf	Zebrafish	CdCl2	112	Altered adult hyperactivity and antioxidant physiology	Ruiter et al. (2016)
4d	0–96 hpf	Zebrafish	Cd3	112	Behavioral alteration, oxidative stress, immunotoxicity	Jin et al. (2015)
5d	0–120 hpf	Zebrafish	CdCl2	560	Induction of mt in olfactory pits and neuromast	Chen et al. (2007)
4d	0–96 hpf	Zebrafish	Cd3	2000	Reduction in neural crest gene expression and hypopigmentation	Zhang et al. (2015)
3d	0–72 hpf	Zebrafish	Cd3	2040	Neuromast damage	Sonnack et al. (2015)
~1d	4–24 hpf	Zebrafish	CdCl2	6893	Hyperpigmentation, reduced retinal ganglion projections, no photoreceptors, reduced neuronal projections	Hen Chow et al. (2009)
~1d	4–24 hpf	Zebrafish	CdCl2	11241	Decreased head size, unclear brain divisions, reduced proneuronal gene expression	Chow et al. (2008)

¹Lowest Observed Effect Concentration;

²parts per billion;

³Species not specified.

In zebrafish, low level (40 ppb) developmental exposure to cadmium results in hyperactivity, decreased otolith size (inner ear gravity sensing biocrystals), and increases in rotational movement¹⁶. While short term (24 – 120 hours) moderate exposures (112 – 560 ppb), as seen in larvae, show increases in antioxidant and detoxifying (cyp19a1b and mt1) gene expression, oxidative stress, immunotoxicity, damage to the olfactory pits and neuromast cells, and a reduction in olfactory-dependent predator response^64–67. An interesting finding from a recently published study revealed that in vivo and in vitro exposure to 112 ppb Cd resulted in anti-estrogenic activity⁶⁴, although the mechanism remains unknown. At higher concentrations (2 ppm), indications of neural crest effects were apparent, including disruptions in neural crest gene expression patterns and hypopigmentation; in addition, neuromast damage, and eye hypoplasia were reported^68,69. Paradoxically, two studies using very high Cd levels (~7 and 11 ppm) found hyperpigmentation, as well as reductions in retinal ganglion projections, optic neuronal projections, a complete absence of photoreceptors, decreased head size, unclear brain divisions, and reduced proneuronal gene expression^70,71. Longer (50 days) exposure to 20 ppb Cd resulted in reduction of olfactory-dependent predator response in juvenile fish, in a manner similar to that observed in the larval studies referenced above^20,62,72, which may reflect equivalent accumulations of cadmium in the three studies despite the differences in Cd levels at the onset of the experiments. Overall, embryonic Cd exposure in zebrafish shows similar responses as in adults, including indicators of oxidative stress but the developing sensory system appears to be particularly sensitive to Cd toxicity.

Lead (Pb)

Lead has received attention in recent years due to contamination of multiple public water systems. The effects of lead on zebrafish have been summarized in a recent review by Lee and Freeman (2014), including discussions on neurodegenerative diseases and the role of Pb in their development, and the use of zebrafish as a model organism⁷³. As such we discuss results published since 2014, which are summarized in Table 3. Unlike Cd studies, the effects of Pb exposure during development have been examined at much lower concentrations, ranging from 10 to ~200 ppb. At lower levels (< 100 ppb), studies show that Pb alters a number of genes associated with nervous system development⁷⁴, including increased GABA gene and protein expression early in development, which decreases after hatching⁷⁵. These changes are possibly associated with decreased neuronal axon length, and reduced activity (hypoactivity)⁷⁶. At levels above 100 ppb, zebrafish exhibit decreased adult learning, and altered color preferences^77,78, with the former persisting for up to three generations after the initial exposure. These findings confirm previous observations regarding interference with axon development, and learning and memory deficits but the underlying mechanisms and windows of susceptibility require further investigation⁷³.

Table 3.

Summary of Zebrafish Studies Measuring Neurological Effects of Lead (Pb) Exposure

Exposure Duration	Age at Exposure	Chemical	LOEC(ppb)	Major Endpoints	Reference
3d	0–72 hpf	Pb(CH3COO)2	10	Gene expression changes in 89 genes associated with nervous system development	Lee and Freeman (2014)
3d	0–72 hpf	Pb(CH3COO)2	10	Altered GABA gene expression	Wirbisky et al. (2014)
6d	0–144 hpf	Pb(CH3COO)2	20	Decreased axon length and decreased locomotion (speed)	Zhu et al. (2016)
~3d	2–72 hpf	Pb(CH3COO)2	100	No change in Alzheimer’s disease-associated gene, sorl1	Lee et al.(2017)
~5d	2–120 hpf	Pb(CH3COO)2	100	Altered color preference (Adults)	Bault et al. (2015)
~2d	2–24 hpf	Lead nitrate (Pb(NO3)2)	207	Decreased Learning (Adults)	Xu et al. (2016)

Methylmercury (MeHg)

Studies investigating the effects of mercury exposure have used both zebrafish and fathead minnows (see Table 4). In adults, these studies have shown that at levels less than 200 ppb, MeHg inhibits membrane adenosine deaminase, and results in mt2 gene induction in the brain but otherwise has minimal impact on other brain transcripts^79–81. Mid-range levels (between 720 to 5500 ppb) show significant Hg accumulation in the brain⁸², delayed hatching, and increased mortality⁸³, and the induction of hyperactive behavior coupled with decreased levels of the neurotransmitters, serotonin and dopamine^84,85. At high doses (10 – 13 ppm), studies show alterations in proteins associated with gap junctions and oxidative phosphorylation, large increases in mt2, and mitochondrial dysfunction^81,82,86.

Table 4.

Summary of Studies Measuring Neurological Effects of Mercury (Hg) Exposure

Exposure Duration	Age of Exposure	Species	Chemical	LOEC(PPB)	Major Endpoints	Reference
1d	Adult	Zebrafish	Mercury chloride (HgCl2)	60	Inhibition of membrane adenosine deaminase	Senger et al. (2010)
54d	Adult	Zebrafish	Methylmercury(MeHg)	80	No change in brain gene expression (qpcr)	Gonzalez et al. (2005)
7d	Adult	Zebrafish	MeHg	119	mt2 gene induction in brain	Gentes et al. (2015)
30d	Adult	Fathead minnow	MeHg	720	Hyperactivity and decreased dopamine	Bridges et al. (2016)
30d	Adult	Fathead minnow	MeHg	870	Hyperactivity & decreased hatching time	Bridges et al. (2016)
56d	Adult	Zebrafish	MeHg	5000	Accumulation in Brain	Amlund et al. (2015)
1d	Adult	Zebrafish	MeHg	5000	Hyperactivity and decreased serotonin	Maximino et al. (2011)
30d	Adult	Fathead minnow	MeHg	5500	Delayed hatching and increased mortality	Bridges et al. (2016)
56d	Adult	Zebrafish	MeHg	10000	Altered proteins associated with gap junction signaling, oxidative phosphorylation, and mitochondrial dysfunction	Rasinger et al. (2017)
56d	Adult	Zebrafish	MeHg	10000	Accumulation in Brain	Amlund et al. (2015)
62d	Adult	Zebrafish	MeHg	11001	mt2 protein increase in brain	Gentes et al. (2015)
49d	Adult	Zebrafish	MeHg	13000	Brain mitochondrial respiration (unaffected)	Bourdineaud et al. (2013)
30d	Adult & Embryo	Fathead minnow	MeHg	720	Hyperactivity and decreased dopamine	Cambier et al. (2012)
~1d	4–24 hpf	Zebrafish	MeHg	3	Adult visual deficit	Weber et al. (2008)
~3d	6 hpf – 72 hpf	Zebrafish	MeHg	10	Decreased neural tube cell proliferation	Hassan et al. (2012)
~3d	5 hpf – 72 hpf	Zebrafish	HgCl2	27	Hyperactivity and mortality	Abu Bakar et al. (2016)
~3d	6 hpf – 72 hpf	Zebrafish	MeHg	50	Delayed hatching	Hassan et al. (2012)
1d	48 hpf – 72 hpf	Zebrafish	MeHg	60	Decrease in head size & alteration of cAMP signaling pathway	Ho et al. (2013)
~3d	6 hpf – 72 hpf	Zebrafish	MeHg	100	Mortality	Hassan et al. (2012)
2d	2–4 dpf	Zebrafish	MeHg	136	Accumulation in photoreceptors in retina and pineal gland	Korbas et al. (2013)
10m	5 dpf	Zebrafish	HgCl2	13576	Accumulation in Brain	Bera et al. (2014)

Studies in zebrafish embryos find that this stage is significantly more sensitive to MeHg with significant molecular, cellular and behavioral effects emerging at much lower levels. For example, embryonic exposure to Hg levels less than 30 ppb results in adult visual deficits,⁸⁷ decreased neural tube cell profileration⁸⁸, hyperactivity, and mortality⁸⁹. Levels above 50 ppb result is significant toxic outcomes including delayed hatching⁸⁸, decreased head size⁹⁰, altered cAMP signaling⁹⁰, and mortality⁸⁸.

CONCLUSION

Research is still needed to expand understanding into the effects of heavy metal exposure on neurodevelopment and neurodegenerative diseases. However, recent advances in the production of transgenic zebrafish lines for neurodevelopment studies, and the use of other aquatic species to study metal toxicity have returned promising results that can be used to understand mechanisms of metal toxicity, and may lead to interventions for exposed populations or new regulatory policies aimed at reducing the levels of heavy metals in the environment. Although still a vexing problem, the current is moving in the right direction and aquatic models are helping navigate the perilous waters of heavy metal toxicity.