This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Thesis: Ph. D., Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Department of Biology; and the Woods Hole Oceanographic Institution), 2019 / Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references. / Harmful algal blooms (HABs) can produce potent neurotoxins that accumulate in seafood and affect human health. One HAB toxin of concern is domoic acid (DomA), a glutamate analog produced by the marine diatom Pseudo-nitzschia spp. Current regulatory limits are designed to prevent acute neurotoxicity in adult humans. However, research shows that low-level exposure during early life can lead to long-term changes in behavior, neural connectivity, and brain morphology. To determine the underlying mechanisms of developmental toxicity, this dissertation used zebrafish as a tool to: i) Establish the developmental window of susceptibility for DomA toxicity, ii) Characterize the behavioral consequences of exposures, and iii) Identify the cellular targets and processes perturbed by DomA. I found that DomA exposure particularly at 2 days post fertilization (dpf) led to altered startle response behavior, myelination defects, and the downregulation of axonal and myelin structural genes. / Using vital dyes and immunolabeling, I assessed DomA-induced alterations in cells required for the startle response. I found no differences in the number of sensory neuromasts or in the sensory cranial ganglia structures that detect the acoustic stimuli. However, the majority of DomA-treated larvae lacked one or both Mauthner cells - hindbrain neurons critical for fast startle responses. DomA-treated larvae also had oligodendrocytes with fewer and shorter myelin sheaths, and appeared to aberrantly myelinate neuronal cell bodies. The loss of the Mauthner neurons and their axons may lead to a cellular environment where oligodendrocytes myelinate neuronal cell bodies in the absence of adequate axonal targets. Indeed, pharmacological treatment that reduced the oligodendrocyte number also led to the reduction in the number of these aberrant, myelinated cell bodies. / These results indicate that exposure to DomA at a particular period in neural development targets specific cell types, disrupts myelination in the spinal cord, and leads to prolonged behavioral deficits. These mechanistic insights support hazard assessments of DomA exposures in humans during critical periods in early development. / "Funding for my research came from the Ocean Ventures Fund, Hill family foundation, Woods Hole Sea grant NA14OAR4170074, and the Woods Hole Center for Oceans and Human Health (COHH), which is jointly funded by the National Institutes of Health (P01ES02192, P01ES028938), and the National Science Foundation (OCE-1314642, OCE-1840381). My funding came from the National Institutes of Health (NIH) P01ES021923-04S1, the Ocean Ridge Initiative Fellowship, the Von Damm Fellowship, and the MIT/WHOI Joint Program Academic Programs Office"--Page 5 / by Jennifer Martinez Panlilio. / Ph. D. / Ph.D. Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Department of Biology; and the Woods Hole Oceanographic Institution)

Domoic acid (DA), a potent neurotoxin that has the potential to cause amnesic shellfish poisoning (ASP), is produced by members of the marine diatom genus Pseudo-nitzschia. Outbreaks of ASP in humans and of DA poisoning in birds and marine mammals have been reported across the United States and Canada since the late 1980's. Pseudo-nitzschia species can be extremely abundant in Florida waters, with densities often exceeding 106 cells/L, and sometimes exceeding 107 cells/L. Based on preliminary data, it is evident that at least nine species of Pseudo-nitzschia are found in Florida coastal waters. At least six of these species are known to produce DA in other parts of the world, and some are morphologically identical to some of the major toxin-producing species in Californian and Canadian waters. Despite the strong presence of Pseudo-nitzschia, there has never been a report of ASP or a DA-related animal mortality event from Florida. Data collected from 2004 to 2011 show maximum Pseudo-nitzschia abundances exceeded 4 x 107 cells/L. Six species of Pseudo-nitzschia were identified from central west and southwest Florida waters via light and electron microscopy. This is the first report of P. micropora from the Gulf of Mexico. Additionally P. calliantha, P. cuspidata, and P. pungens were identified as producers of DA in Florida coastal waters; although cell quotas of DA were low. Low levels of DA were detected in about one third of the water samples analyzed and DA concentrations measured in the majority of shellfish from the study area were at least an order of magnitude below the regulatory limit of 20 µg/g, suggesting that Pseudo-nitzschia currently poses little threat to human health in Florida. However, DA production in Pseudo-nitzschia species has been shown to be variable and dependent on nutrient conditions, indicating that the potential for DA-related events to occur in Florida warrants further investigation.

Dans une ferme d'élevage de bar (Dicentrarchus labrax) du sud de la mer du Nord, de fortes mortalités de bar coïncident régulièrement avec l'efflorescence phytoplanctonique printanière. Le rôle du phytoplancton dans ces mortalités a donc été étudié : un suivi saisonnier (février-novembre 2012) a permis de définir les communautés phytoplanctoniques en présence et, la diversité et la toxicité du genre Pseudo-nitzschia. Ainsi, trois espèces potentiellement toxiques ont été identifiées (P. delicatissima, P. pungens, P. fraudulenta) en association avec des concentrations élevées d'acide domoïque (jusqu'à 229 pg. mL-¹). Au cours d'une expérience d'exposition (45 jours), les effets délétères de P. delicatissima sur les juvéniles de bar ont été étudiés. Si un stress d'exposition a été observé via la surproduction de mucus par l'épithélium branchial, cela n'a pas eu d'incidence sur la condition ni la physiologie des poissons. Les mortalités seraient donc davantage liées à un effet de communautés. Cette hypothèse a été testée en utilisant le pouvoir de filtration des moules (Mytilis edulis) en amont des bassins d'élevage. Cela a permis de limiter l'ampleur de l'efflorescence phytoplanctonique printanière. En conséquence, les poissons élevés dans l'eau filtrée avaient de meilleures conditions, croissance et rapport ARN:ADN que ceux élevés dans l'eau de mer non filtrée. Ce travail souligne la nécessité de généraliser le suivi des communautés phytoplanctoniques afin d'identifier les espèces délétères et leur dynamique et de développer des outils de mitigation permettant d'atténuer l'impact des efflorescences phytoplanctoniques sur l'aquaculture. / For several years, mass mortalities of farmed sea bass (Dicentrarchus labrax) have coincided with phytoplankton spring blooms in the southern North Sea. Since these mortalities could not be explained by classical finfish diseases, phytoplankton noxious effects have been suspected and investigated. A seasonal survey allowed the identification of potentially deleterious phytoplankton species giving particular attention to the Pseudo-nitzschia genus. Three potentially toxic Pseudo-nitzschia species were identified (P. delicatissima, P. pungens, P. fraudulenta) and their presence was related to both domoic acid concentrations and phytoplankton communities. P. delicatissima being dominant over spring and presenting toxic and physical features compatible with fish mortality, a laboratory exposure experiment was carried out. Although gills irritations (mucus overproduction) revealed an exposure stress, no effect on sea bass condition nor on physiological performances was demonstrated. This suggest that phytoplankton community as a whole rather than single species should be involved in fish mortalities. This third hypothesis was tested using mussels (Mytilus edulis) as seawater filters upstream from the rearing tanks to dampen the phytoplankton spring bloom and estimate its impact on fish. Indeed, fish had better condition , growth and RNA:DNA ratio when reared in filtered seawater than in natural seawater. This work highlights the need to develop phytoplankton monitoring in fish farms so as to identify potentially deleterious species and understand their dynamics. It also demonstrates that new mitigation tools should be developed to prevent phytoplankton impacts on farmed fish.

Over the past 60 years, a large number of selective neurotoxins were discovered and developed, making it possible to animal-model a broad range of human neuropsychiatric and neurodevelopmental disorders. In this paper, we highlight those neurotoxins that are most commonly used as neuroteratologic agents, to either produce lifelong destruction of neurons of a particular phenotype, or a group of neurons linked by a specific class of transporter proteins (i.e., dopamine transporter) or body of receptors for a specific neurotransmitter (i.e., NMDA class of glutamate receptors). Actions of a range of neurotoxins are described: 6-hydroxydopamine (6-OHDA), 6-hydroxydopa, DSP-4, MPTP, methamphetamine, IgG-saporin, domoate, NMDA receptor antagonists, and valproate. Their neuroteratologic features are outlined, as well as those of nerve growth factor, epidermal growth factor, and that of stress. The value of each of these neurotoxins in animal modeling of human neurologic, neurodegenerative, and neuropsychiatric disorders is discussed in terms of the respective value as well as limitations of the derived animal model. Neuroteratologic agents have proven to be of immense importance for understanding how associated neural systems in human neural disorders may be better targeted by new therapeutic agents.

Neurotoxlns represent unique chemical tools, providing a means to 1) gain insight into cellular mechanisms of apopotosis and necrosis, 2) achieve a morphological template for studies otherwise unattainable, 3) specifically produce a singular phenotype of denervation, and 4) provide the starting point to delve into processes and mechanisms of nerve regeneration and sprouting. There are many other notable uses of neurotoxins in neuroscience research, and ever more being discovered each year. The objective of this review paper is to highlight the broad areas of neuroscience in which neurotoxins and neurotoxicity mechanism come into play. This shifts the focus away from neurotoxins per se, and onto the major problems under study today. Neurotoxins broadly defined are used to explore neurodegenerative disorders, psychiatric disorders and substance use disorders. Neurotoxic mechanisms relating to protein aggregates are indigenous to Alzheimer disease, Parkinson's disease. NeuroAIDS is a disorder in which microglia and macrophages have enormous import. The gap between the immune system and nervous system has been bridged, as neuroinflammation is now considered to be part of the neurodegenerative process. Related mechanisms now arise in the process of neurogenesis. Accordingly, the entire spectrum of neuroscience is within the purview of neurotoxins and neurotoxicity mechanisms. Highlights on discoveries in the areas noted, and on selective neurotoxins, are included, mainly from the past 2 to 3 years.

The era of selective neurotoxins arose predominately in the 1960s with the discovery of the norepinephrine (NE) isomer 6-hydroxydopamine (6-OHDA), which selectively destroyed noradrenergic sympathetic nerves in rats. A series of similarly selective neurotoxins were later discovered, having high affinity for the transporter site on nerves and thus being accumulated and able to disrupt vital intraneuronal processes, to lead to cell death. The Trojan Horse botulinum neurotoxins (BoNT) and tetanus toxin bind to glycoproteins on the neuronal plasma membrane, then these stealth neurotoxins are taken inside respective cholinergic or glycinergic nerves, producing months-long functional inactivation but without overtly destroying those nerves. The mitochondrial complex I inhibitor rotenone, while lacking total specificity, still destroys dopaminergic nerves with some selectivity; and importantly, results in the neural accumulation of synuclein-to model Parkinson’s disease (PD) in animals. Other neurotoxins target specific subtypes of glutamate receptors and produce excitotoxicity in nerves with that receptor population. The dopamine D2 receptor agonist quinpirole, termed a selective neurotoxin, produces a behavioral state replicating some of the notable features of schizophrenia, but without overtly destroying nerves. These processes, mechanisms or treatment-outcomes account for the means by which neurotoxins are classified as such, and represent some of the means by which neurotoxins as a group are able to destroy or functionally inactivate nerves; or replicate an altered neurological state. Selective neurotoxins have proven to be important in gaining insight into biochemical processes and mechanisms responsible for survival or demise of a nerve. Selective neurotoxins are useful also for animal modeling of human neural disorders such as PD, Alzheimer disease, attention-deficit hyperactivity disorder (ADHD), Lesch-Nyhan disease, tardive dyskinesia, schizophrenia and others. The importance of neurotoxins in neuroscience will continue to be ever more important as even newer neurotoxins are discovered.

There has been an awareness of nerve poisons from ancient times. At the dawn of the twentieth century, the actions and mechanisms of these poisons were uncovered by modern physiological and biochemical experimentation. However, the era of selective neurotoxins began with the pioneering studies of R. Levi-Montalcini through her studies of the neurotrophin "nerve growth factor" (NGF), a protein promoting growth and development of sensory and sympathetic noradrenergic nerves. An antibody to NGF, namely, anti-NGF - developed in the 1950s in a collaboration with S. Cohen - was shown to produce an "immunosympathectomy" and virtual lifelong sympathetic denervation. These Nobel Laureates thus developed and characterized the first identifiable selective neurotoxin. Other selective neurotoxins were soon discovered, and the compendium of selective neurotoxins continues to grow, so that today there are numerous selective neurotoxins, with the potential to destroy or produce dysfunction of a variety of phenotypic nerves. Selective neurotoxins are of value because of their ability to selectively destroy or disable a common group of nerves possessing (1) a particular neural transporter, (2) a unique set of enzymes or vesicular transporter, (3) a specific type of receptor or (4) membranous protein, or (5) other uniqueness. The era of selective neurotoxins has developed to such an extent that the very definition of a "selective" neurotoxin has warped. For example, (1) N-methyl-D- aspartate receptor (NMDA-R) antagonists, considered to be neuroprotectants by virtue of their prevention of excitotoxicity from glutamate receptor agonists, actually lead to the demise of populations of neurons with NMDA receptors, when administered during ontogenetic development. The mere lack of natural excitation of this nerve population, consequent to NMDA-R block, sends a message that these nerves are redundant - and an apoptotic cascade is set in motion to eliminate these nerves. (2) The rodenticide rotenone, a global cytotoxin that acts mainly to inhibit complex I in the respiratory transport chain, is now used in low dose over a period of weeks to months to produce relatively selective destruction of substantia nigra dopaminergic nerves and promote alpha-synuclein deposition in brain to thus model Parkinson's disease. Similarly, (3) glial toxins, affecting oligodendrocytes or other satellite cells, can lead to the damage or dysfunction of identifiable groups of neurons. Consequently, these toxins might also be considered as "selective neurotoxins," despite the fact that the targeted cell is nonneuronal. Likewise, (4) the dopamine D2-receptor agonist quinpirole, administered daily for a week or more, leads to development of D2-receptor supersensitivity - exaggerated responses to the D2-receptor agonist, an effect persisting lifelong. Thus, neuroprotectants can become "selective" neurotoxins; nonspecific cytotoxins can become classified as "selective" neurotoxins; and receptor agonists, under defined dosing conditions, can supersensitize and thus be classified as "selective" neurotoxins. More examples will be uncovered as the area of selective neurotoxins expands. The description and characterization of selective neurotoxins, with unmasking of their mechanisms of action, have led to a level of understanding of neuronal activity and reactivity that could not be understood by conventional physiological observations. This chapter will be useful as an introduction to the scope of the field of selective neurotoxins and provide insight for in-depth analysis in later chapters with full descriptions of selective neurotoxins.