A Novel Antibody-Based Biomarker for Chronic Algal Toxin Exposure and Sub-Acute Neurotoxicity

The neurotoxic amino acid, domoic acid (DA), is naturally produced by marine phytoplankton and presents a significant threat to the health of marine mammals, seabirds and humans via transfer of the toxin through the foodweb. In humans, acute exposure causes a neurotoxic illness known as amnesic shellfish poisoning characterized by seizures, memory loss, coma and death. Regular monitoring for high DA levels in edible shellfish tissues has been effective in protecting human consumers from acute DA exposure. However, chronic low-level DA exposure remains a concern, particularly in coastal and tribal communities that subsistence harvest shellfish known to contain low levels of the toxin. Domoic acid exposure via consumption of planktivorous fish also has a profound health impact on California sea lions (Zalophus californianus) affecting hundreds of animals yearly. Due to increasing algal toxin exposure threats globally, there is a critical need for reliable diagnostic tests for assessing chronic DA exposure in humans and wildlife. Here we report the discovery of a novel DA-specific antibody response that is a signature of chronic low-level exposure identified initially in a zebrafish exposure model and confirmed in naturally exposed wild sea lions. Additionally, we found that chronic exposure in zebrafish caused increased neurologic sensitivity to DA, revealing that repetitive exposure to DA well below the threshold for acute behavioral toxicity has underlying neurotoxic consequences. The discovery that chronic exposure to low levels of a small, water-soluble single amino acid triggers a detectable antibody response is surprising and has profound implications for the development of diagnostic tests for exposure to other pervasive environmental toxins

Neurological impacts of prolonged asymptomatic exposure to the marine neurotoxin domoic acid

Domoic acid is an algal-derived neurotoxin that contaminates seafood during harmful algal blooms. It causes excitotoxicity in the vertebrate central nervous system by over-stimulating neurons; in high doses, this can cause seizures, brain lesions, and death. At low doses, however – those causing no outward signs of excitotoxicity – it is unclear whether domoic acid exposure could have potentially damaging neurological effects. This is especially relevant for human exposures, as we are protected from high toxin levels by seafood monitoring and regulatory efforts, but can still ingest domoic acid at concentrations below the regulatory limit (20 µg domoic acid per g wet shellfish weight). To address whether chronic exposure to these low-levels of domoic acid negatively affects neurological health and function, I: 1) examined changes in gene expression and cellular energetics in the brains of adult zebrafish chronically exposed to asymptomatic domoic acid; 2) assessed the effect of low-dose (i.e., non-cytotoxic) domoic acid exposure on the electrophysiological activity and connectivity of neural networks in organotypic mouse brain slice cultures in vitro; and 3) examined the brains of adult mice chronically exposed to asymptomatic domoic acid for signs of histopathology and disruption to a subtype of parvalbumin-positive inhibitory neurons. I found that prolonged domoic acid exposure significantly affected gene transcription and impaired mitochondrial function in zebrafish brains, and altered neuronal network activity and connectivity in mouse brain slice cultures, all in the absence of overt symptoms, gross histopathology, and neuron death/injury. Further, chronic asymptomatic domoic acid exposures that led to transient deficits in learning and memory in a mouse model did not produce measurable effects on hippocampal inflammation, cell/neuron numbers, or parvalbumin-positive staining intensity. These results indicate that chronic domoic acid exposure may have sub-clinical effects that can go undetected and may impact neurological function; further study is needed to better inform human health risk assessments

Neurological impacts of prolonged asymptomatic exposure to the marine neurotoxin domoic acid

Domoic acid is an algal-derived neurotoxin that contaminates seafood during harmful algal blooms. It causes excitotoxicity in the vertebrate central nervous system by over-stimulating neurons; in high doses, this can cause seizures, brain lesions, and death. At low doses, however – those causing no outward signs of excitotoxicity – it is unclear whether domoic acid exposure could have potentially damaging neurological effects. This is especially relevant for human exposures, as we are protected from high toxin levels by seafood monitoring and regulatory efforts, but can still ingest domoic acid at concentrations below the regulatory limit (20 µg domoic acid per g wet shellfish weight). To address whether chronic exposure to these low-levels of domoic acid negatively affects neurological health and function, I: 1) examined changes in gene expression and cellular energetics in the brains of adult zebrafish chronically exposed to asymptomatic domoic acid; 2) assessed the effect of low-dose (i.e., non-cytotoxic) domoic acid exposure on the electrophysiological activity and connectivity of neural networks in organotypic mouse brain slice cultures in vitro; and 3) examined the brains of adult mice chronically exposed to asymptomatic domoic acid for signs of histopathology and disruption to a subtype of parvalbumin-positive inhibitory neurons. I found that prolonged domoic acid exposure significantly affected gene transcription and impaired mitochondrial function in zebrafish brains, and altered neuronal network activity and connectivity in mouse brain slice cultures, all in the absence of overt symptoms, gross histopathology, and neuron death/injury. Further, chronic asymptomatic domoic acid exposures that led to transient deficits in learning and memory in a mouse model did not produce measurable effects on hippocampal inflammation, cell/neuron numbers, or parvalbumin-positive staining intensity. These results indicate that chronic domoic acid exposure may have sub-clinical effects that can go undetected and may impact neurological function; further study is needed to better inform human health risk assessments

Multiplex networks of cortical and hippocampal neurons revealed at different timescales.

Recent studies have emphasized the importance of multiplex networks--interdependent networks with shared nodes and different types of connections--in systems primarily outside of neuroscience. Though the multiplex properties of networks are frequently not considered, most networks are actually multiplex networks and the multiplex specific features of networks can greatly affect network behavior (e.g. fault tolerance). Thus, the study of networks of neurons could potentially be greatly enhanced using a multiplex perspective. Given the wide range of temporally dependent rhythms and phenomena present in neural systems, we chose to examine multiplex networks of individual neurons with time scale dependent connections. To study these networks, we used transfer entropy--an information theoretic quantity that can be used to measure linear and nonlinear interactions--to systematically measure the connectivity between individual neurons at different time scales in cortical and hippocampal slice cultures. We recorded the spiking activity of almost 12,000 neurons across 60 tissue samples using a 512-electrode array with 60 micrometer inter-electrode spacing and 50 microsecond temporal resolution. To the best of our knowledge, this preparation and recording method represents a superior combination of number of recorded neurons and temporal and spatial recording resolutions to any currently available in vivo system. We found that highly connected neurons ("hubs") were localized to certain time scales, which, we hypothesize, increases the fault tolerance of the network. Conversely, a large proportion of non-hub neurons were not localized to certain time scales. In addition, we found that long and short time scale connectivity was uncorrelated. Finally, we found that long time scale networks were significantly less modular and more disassortative than short time scale networks in both tissue types. As far as we are aware, this analysis represents the first systematic study of temporally dependent multiplex networks among individual neurons

Chronic low-level exposure to the common seafood toxin domoic acid causes cognitive deficits in mice

The consumption of one meal of seafood containing domoic acid (DA) at levels high enough to induce seizures can cause gross histopathological lesions in hippocampal regions of the brain and permanent memory loss in humans and marine mammals. Seafood regulatory limits have been set at 20mgDA/kg shellfish to protect human consumers from symptomatic acute exposure, but the effects of repetitive low-level asymptomatic exposure remain a critical knowledge gap. Recreational and Tribal-subsistence shellfish harvesters are known to regularly consume low levels of DA. The aim of this study was to determine if chronic low-level DA exposure, at doses below those that cause overt signs of neurotoxicity, has quantifiable impacts on cognitive function. To this end, female C57BL/6NJ mice were exposed to asymptomatic doses of DA (≈0.75mg/kg) or vehicle once a week for several months. Spatial learning and memory were tested in a radial water maze paradigm at one, six and 25 weeks of exposure, after a nine-week recovery period following cessation of exposure, and at three old age time points (18, 24 and 28 months old). Mice from select time points were also tested for activity levels in a novel cage environment using a photobeam activity system. Chronic low-level DA exposure caused significant spatial learning impairment and hyperactivity after 25 weeks of exposure in the absence of visible histopathological lesions in hippocampal regions of the brain. These cognitive effects were reversible after a nine-week recovery period with no toxin exposure and recovery was sustained into old age. These findings identify a new potential health risk of chronic low-level exposure in a mammalian model. Unlike the permanent cognitive impacts of acute exposure, the chronic low-level effects observed in this study were reversible suggesting that these deficits could potentially be managed through cessation of exposure if they also occur in human seafood consumers

Hub sharing was limited to adjacent time scales.

<p>(<b>A</b>) We classified each neuron as a hub, non-hub, or unconnected neuron at each time scale. A neuron was considered to be a shared hub or shared non-hub for two time scales if its status as a hub or non-hub was consistent across those time scales. Hubs were defined using a degree threshold set by the likelihood to have a given number of connections in a random network (0.05 in this illustrative diagram and 10⁻⁴ in the full analysis). (<b>B</b>) We calculated the amount of hub and non-hub sharing (see <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115764#s4" target="_blank">Materials and Methods</a></i>) for each pair of time scales and grouped the results into neighboring (4 or less) and distant (greater than 4) time scales. <i><u>We found that hubs were only shared at a significant level for neighboring time scales, while non-hubs were broadly shared across all time scales</u></i> (multiple comparisons correct Mann-Whitney Test (1, 2, and 3 dots: p<0.05, 0.01, and 0.001 respectively), error bar: standard error of the mean). For each data set, we subtracted the mean sharing values for 500 trials with neuron identities randomized and neuron hub, non-hub, or unconnected status held constant. This null model approximates the amount of sharing expected based only on the number of hubs, non-hubs, and unconnected neurons in the data set, as well as the effect of ignoring the multiplex properties of the networks and considering the time scales to be truly independent networks. We also calculated the mean sharing value of (<b>C</b>) hubs and (<b>E</b>) non-hubs across each pair of time scales for cortical and hippocampal networks. In (B), neighboring time scale pairs are up and to the left of the white line, while distant time scale pairs are down and to the right of the white line. (<b>D and F</b>) Finally, we calculated the multiple comparisons corrected Mann-Whitney Test p-values between sharing results from data and sharing results from the null model.</p

Binning structure for short time scales on example spike trains.

<p>Note that the time scales overlapped to some degree to capture interactions with all delays and that time scales greater than 1 possessed delays to prevent short time scale interactions from influencing long time scale measurements.</p

Hippocampal structures were preserved throughout culturing. Photographs of cortico-hippocampal organotypic cultures.

<p>(<b>A</b>) A bright field image of an example organotypic culture at DIV1. The hippocampal structure is visible without staining. Blue arrows indicate the location of the edge of the recording array. (<b>B</b>) NeuN staining of the culture after data taking and tissue fixation at DIV16. There are missing neurons in CA3 as consistent with a previous report <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115764#pone.0115764-Zimmer1" target="_blank">[111]</a>, but the overall layer structure is well conserved. (<b>C</b>) Overlaid photograph of A and B. Positions and dimensions of the hippocampal structures are well conserved during the incubation period. (<b>D</b>) Overlaid photograph of B, the outline of the array (yellow rectangle), and the estimated locations of the recorded neurons. Light blue circles are manually identified hippocampal neurons and red circles are neurons recorded outside the hippocampal structure. Locations of the recorded neurons match with the granule cell layer and the cell body layer. For complete details on culture preparation, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115764#pone.0115764-Ito2" target="_blank">[48]</a>.</p