The excitotoxin domoic acid (DOM) acts at both kainic acid (KA)- and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-sensitive glutamate receptors. Clinical reports suggest that elderly people are hypersensitive to the neurological effects of DOM intoxication. Young, but not aged hippocampal slices which have been preconditioned with low concentrations of DOM or KA exhibit an acute �tolerance� to subsequent high doses of DOM or KA; application of the selective AMPA agonist fluorowillardiine (FW) fails to induce tolerance to excitotoxins. The aim of this study was to further investigate the molecular mechanism of tolerance induction in vitro, and to examine the ability of compounds to cross-condition against excitotoxic insult. In addition, in vivo techniques were used to explore the age-related susceptibility to the neurological effects of DOM and acute in vivo tolerance induction. Here we show that low doses of �classical� ionotropic kainate receptor agonists and AMPA/kainate receptor antagonists act as net inverse agonists at G-protein coupled receptors, reducing constitutive GTPase activity by up to 73% in the young hippocampus. Further evidence that inverse agonist activity at G-protein coupled receptors is responsible for acute in vitro tolerance induction by kainate receptor agonists and antagonists was also identified because preconditioning with the AMPA receptor antagonist GYKI-52466 significantly inhibited KA-induced population spike suppression in in vitro hippocampal brain slices from both Sprague-Dawley and Wistar rats. The broad-spectrum protein kinase inhibitor H-7 partially blocked tolerance induction when preconditioning occurs in the presence of suggesting that protein kinases are one of the downstream effectors of this phenomenon. Tolerance-inducing compounds are also capable of cross-conditioning against the effects of other excitotoxins; with 250 nM FW suppressed population spike area by only 62.8 � 10.0% at 30 minutes following a 500 nM KA preconditioning dose, compared to almost complete spike suppression within twenty minutes in naive hippocampal brain slices. In vivo experiments indicated that despite aged animals exhibiting significantly higher cumulative behavioural scores in response to i.p. DOM (1 mg kg⁻�; young = 102 � 9, aged = 179 � 19; P < 0.01) in response to DOM after two hours), and that this age-related supersensitivity is due to impaired renal clearance (young serum DOM = 41.5 � 30.3 ng ml⁻�, aged = 813.3 � 804.4 ng ml⁻� following administration of 1 mg kg⁻� DOM after 2.5 hours earlier). Tolerance to high doses of DOM was induced within a matter of minutes following i.p. preconditioning by low dose DOM in vivo. This was evidenced by severe seizure manifestations being almost absent in both young and aged animals, despite occurring frequently in naive animals. Therefore, this study concludes that tolerance is induced by kainate receptor ligands in vitro and in vivo within a matter of minutes, and is the result of a reduction in the turnover of G-protein coupled receptors and protein kinase activation. In addition, the increased sensitivity of aged rats to in vivo DOM is a result of elevated serum DOM concentrations most likely resulting from impaired renal clearance.

The excitotoxin domoic acid (DOM) acts at both kainic acid (KA)- and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-sensitive glutamate receptors. Clinical reports suggest that elderly people are hypersensitive to the neurological effects of DOM intoxication. Young, but not aged hippocampal slices which have been preconditioned with low concentrations of DOM or KA exhibit an acute �tolerance� to subsequent high doses of DOM or KA; application of the selective AMPA agonist fluorowillardiine (FW) fails to induce tolerance to excitotoxins. The aim of this study was to further investigate the molecular mechanism of tolerance induction in vitro, and to examine the ability of compounds to cross-condition against excitotoxic insult. In addition, in vivo techniques were used to explore the age-related susceptibility to the neurological effects of DOM and acute in vivo tolerance induction. Here we show that low doses of �classical� ionotropic kainate receptor agonists and AMPA/kainate receptor antagonists act as net inverse agonists at G-protein coupled receptors, reducing constitutive GTPase activity by up to 73% in the young hippocampus. Further evidence that inverse agonist activity at G-protein coupled receptors is responsible for acute in vitro tolerance induction by kainate receptor agonists and antagonists was also identified because preconditioning with the AMPA receptor antagonist GYKI-52466 significantly inhibited KA-induced population spike suppression in in vitro hippocampal brain slices from both Sprague-Dawley and Wistar rats. The broad-spectrum protein kinase inhibitor H-7 partially blocked tolerance induction when preconditioning occurs in the presence of suggesting that protein kinases are one of the downstream effectors of this phenomenon. Tolerance-inducing compounds are also capable of cross-conditioning against the effects of other excitotoxins; with 250 nM FW suppressed population spike area by only 62.8 � 10.0% at 30 minutes following a 500 nM KA preconditioning dose, compared to almost complete spike suppression within twenty minutes in naive hippocampal brain slices. In vivo experiments indicated that despite aged animals exhibiting significantly higher cumulative behavioural scores in response to i.p. DOM (1 mg kg⁻�; young = 102 � 9, aged = 179 � 19; P < 0.01) in response to DOM after two hours), and that this age-related supersensitivity is due to impaired renal clearance (young serum DOM = 41.5 � 30.3 ng ml⁻�, aged = 813.3 � 804.4 ng ml⁻� following administration of 1 mg kg⁻� DOM after 2.5 hours earlier). Tolerance to high doses of DOM was induced within a matter of minutes following i.p. preconditioning by low dose DOM in vivo. This was evidenced by severe seizure manifestations being almost absent in both young and aged animals, despite occurring frequently in naive animals. Therefore, this study concludes that tolerance is induced by kainate receptor ligands in vitro and in vivo within a matter of minutes, and is the result of a reduction in the turnover of G-protein coupled receptors and protein kinase activation. In addition, the increased sensitivity of aged rats to in vivo DOM is a result of elevated serum DOM concentrations most likely resulting from impaired renal clearance.

Cardiovascular pathology is seen in both animals and humans after domoic acid intoxication. Whether this damage is direct (i.e., cardiotoxic) or indirect (i.e., CNS/autonomic seizures) is not known. We have previously shown that acute in vitro domoic acid (0.05-0.25[mu]M; 10 min) treatment of isolated cardiac mitochondria compromises mitochondrial FADH and NAD⁺-linked respiratory control and mitochondrial energetics. Domoic acid was shown to traverse and bind the cellular membrane of H9c2 cardiac myoblasts. However it did not compromise cellular viability as assessed using cell quantification or lactate dehydrogenase leakage assays. Exposure of intact H9c2 cells to domoic acid only resulted in complex II-III activity impairment and assessment of reactive oxygen species (superoxide and hydrogen peroxide) production in both isolated cardiac mitochondria and H9c2 cardiomyocytes failed to show any significant differences following exposure to domoic acid. Acute ex vivo domoic acid treatment of an isolated myocardium in Langendorff perfusion mode failed to result in cardiac haemodynamic dysfunction, however there appeared to be small but significant decrease in mitochondrial oxygen utilization. The absence of any substantial damage to intact cardiomyocytes and isolated myocardium suggested that domoic acid does not have a direct toxicological effect on cardiac energetics. We therefore investigated the possibility that cardiovascular pathology is an indirect consequence of autonomic seizure activity. Domoic acid was administered intraperitoneally or intrahippocampally and the development of cardiac pathologies was assessed and compared. Sprague-Dawley rats receiving either i.p. or i.h. domoic acid were assessed behaviourally and shown to reach similar levels in their cumulative seizure scores. Assessment of the cardiac haemodynamics (LVDP, dP/dt, heart rate and coronary flow) revealed a significant time-dependent decrease in function at 1, 3, 7 & 14-days post-i.p. and 7 & 14-days post-i.h. domoic acid administration. Measurement of ventricular mitochondrial oxygen utilization revealed a similar time-dependent decrease in respiratory control, which appeared to be associated with increased proton leakage, shown by an increase in state-4 respiration rate (P<0.01). Assessment of the mitochondrial electron transport chain (complexes I-V) and the mitochondrial marker of integrity, citrate synthase, showed marked time-dependent impairment in both models of domoic acid -induced seizures. Oxidative stress did play a small role in the myocardial damage as indicated by the small decrease in aconitase activity (P<0.05). Plasma IL-1α, IL-1β and TNF-α levels were significantly increased from 3-days post seizures. Haematoxylin & Eosin staining of ventricular sections revealed the formation of contraction bands, inflammation and oedema, confirming a structural pathology. Cardiac damage did not differ between i.p. and i.h. animals, suggesting cardiac damage following domoic acid results from CNS autonomic seizures and resultant sympathetic storm. This thesis has demonstrated, for the first time, that the cardiac pathology seen following domoic acid exposure is most likely to be a result of CNS activation and resultant seizure episodes, and is not a consequence of the direct interaction between domoic acid and the myocardium. We have also demonstrated for the first time, that seizure episodes result in chronic cardiac dysfunction and a structural pathology which is similar, but not identical to that seen following isoprotenerol administration in vivo.

Thesis (Ph.D.)-University of California, Santa Cruz, 2007. / Includes bibliographical references. Also available online. Restricted to UC campuses.

Master of Science / Food Science Institute / Curtis Kastner / Harmful Algal Blooms (HABs) including Cyanobacteria and other toxic marine biota are responsible for similar harmful effects on human health, food safety, ecosystem maintenance, economic losses and liability issues for aquaculture farms as well as the food industry. Detection, monitoring and mitigation are all key factors in decreasing the deleterious effects of these toxic algal blooms. Harmful algal blooms can manifest toxic effects on a number of facets of animal physiology, elicit noxious taste and odor events and cause mass fish as well as animal kills. Such blooms can adversely impact the perception of the efficacy and safety of the food industry, water utilities, the quality of aquaculture and land farming products, as well as cause ripple effects experienced by coastal communities. HABs can adversely impact coastal areas and other areas reliant on local aquatic ecosystems through the loss of revenues experienced by local restaurants, food manufacturers as well as seafood harvesting/processing plants; loss of tourism revenue, decreased property values and a fundamental shift in the lives of those that are reliant upon those industries for their quality of life. This paper discusses Cyanobacteria, macroalgae, HABs, Cyanobacteria toxins, mitigation of HAB populations and their products as well as the ramifications this burgeoning threat to aquatic/ landlocked communities including challenges these toxic algae pose to the field of food science and the economy.

This work investigates, for the first time, the Pseudo-nitzschia (PN) dynamics in the western English Channel (L4) and the environmental factors impacting on domoic acid (DA) production in these waters. This is combined with laboratory studies examining key environmental factors and the multifactorial impact of multiple macronutrient and micronutrient availability on PN growth and DA production. An LC-MS method was established, optimised, and compared with ELISA for the accurate and reproducible extraction and determination of particulate and dissolved DA. The method was used to measure the seasonal variation in DA at L4 during 2009 and this was compared to PN seasonal abundance and diversity. Three groups a P. delicatissima-group, a P. seriata-group, and a P. pungens/multiseries-group were identified and were found to have different ecological distributions with the latter two groups significantly correlating with DA concentration. Macronutrients, in combination with other environmental factors, were found to influence PN populations at L4. Multifactorial laboratory culture experiments investigating the availability of nitrate, phosphate, and silicate, confirmed that the interrelatedness of all these nutrients significantly affected the growth, decline, and DA production of P. multiseries, and highlight the importance of both phosphate and silicate availability for DA production. When the impacts of both macronutrient (phosphate and silicate) and micronutrient (iron and copper) availability were investigated, limited growth and DA production was observed in P. multiseries cultures. Results revealed the complexity and interrelationship of factors affecting both PN growth and DA production. Furthermore, molecular methods were developed to elucidate the PN species present from 2009 Lugol’s-preserved L4 samples. DNA was successfully extracted and amplified from these samples which had been stored for up to 2 years. Initial sequence analysis identified the rbcL DNA marker as an informative site for future work with a number of L4 sequences closely relating to different Pseudo-nitzschia spp.

Thesis (M.S.)--University of North Carolina at Wilmington, 2006. / Includes bibliographical references (leaves: 36-40)

Domoic acid (DA) is a neurotoxin produced by the harmful algae Pseudo-nitzschia that has been directly linked to mass stranding events of the California Sea Lion (CSL). The purpose of this paper is to review the anthropogenic influence on the production of this neurotoxin and examine how human activities are impacting this marine mammal species. A comprehensive and interdisciplinary literature review was conducted to evaluate the future sustainability of the CSL population. It was found that while Pseudo-nitzschia bloom developments are vulnerable to anthropogenic influences, the incontestable existence of natural contributing factors adds a certain complexity to the determination of causalities and the development of solutions. Strong evidence exists to show that DA can cause major and irreversible neurological damage in CSLs. Rehabilitation of DA-impacted CSLs is a polarizing issue in the U.S. and presents interesting implications for sustainable development. While the CSL population is currently healthy and plenteous, the strong abundance of future uncertainties warrants concern. A balance must be found between the involving social, economic, and environmental factors to ensure a promising future for the CSL species.

Introduction California sea lion (Zalophus californianus) health is severely compromised by domoic acid toxicosis, which occurs in high levels during harmful algal blooms of Pseudonitzschia australis along the coast of California. Current diagnostic protocols are often inconclusive due to a 2-48 hour window of detectability within the urinary, circulatory, and gastric systems (Cook, et al. 2011 and Monte, Pers Comm, 2012). Past studies suggest that Z. californianus, with domoic acid toxicosis, commonly display abnormal behaviors (Goldstein, et al. 2008). However, many of these abnormal behaviors are also associated with other diagnoses and are therefore unreliable as diagnostic indicators. This study fills in a knowledge gap relating to abnormal behavior types and their correlation to domoic acid toxicosis and helps solve the problem of current, inconclusive, diagnostic protocols. In this study, my objectives were to identify abnormal behaviors correlated to domoic acid toxicosis, create a diagnostic ethogram, determine the applicability of the method in the field, and determine the applicability of triage based on the relationship between abnormal behaviors and domoic acid levels. Methods I conducted focal animal continuous scans (continuous observation of a single animal at a time, for a set period) with continuous data entry, on animals admitted to the Marine Mammal Center (main study location during 2011-2013) and the Marine Mammal Care Center (comparison location, 2013). I conducted my observations from behind a blind to prevent both human habituation and behavioral influence of the observer. Observations lasted between 10-15 minutes (10 minutes per pen in 2011, 15 minutes per animal in 2012-2013). Subjects were selected based on an admit date no later than 7 days from the observation date. I conducted focal animal continuous scans at Pier 39, a haul out location, in the San Francisco Bay. Animals included in the study had identifying marks or were isolated from other animals (making them easy to identify). I observed animals once per observation day with a total observation period not exceeding 15 minutes per animal. I logged domoic acid levels in feces, urine, and serum (collected by veterinary staff and analyzed with liquid chromatography and bioassays for the presence of domoic acid). I then compared these results to the types and severity of abnormal behaviors displayed by the domoic acid toxicosis sample. Results Results from data collected at the Marine Mammal Center suggest that head weaving (Wilcoxon, p Results from the Pier 39 study suggest that behavioral criteria may be applicable for ruling out domoic acid toxicosis in groups of animals. However, I did not test the method during times of harmful algal blooms. Therefore, the applicability of the method for use as a diagnostic tool in the field is unknown and further research is required. Results for the triage study were inconclusive. The number of animals that tested positive for domoic acid was small and not suitable for statistical analysis. I suggest further research into triage abilities. Conclusion Based on the results of these studies, I can conclude that behavioral analysis offers a reliable diagnostic tool for rescued Z. californianus. Practitioners can use behavioral diagnostic criteria with confidence for the diagnosis of domoic acid toxicosis in Z. californianus.

Chez les diatomées du genre Pseudo-nitzschia, certaines espèces produisent une neurotoxine, l’acide domoïque responsable chez l’homme d’intoxications amnésiantes suite à la consommation de mollusques contaminés. En Baie de Seine, la variabilité interannuelle de ces efflorescences toxiques, qui provoquent certaines années des fermetures de zones de pêche à la coquille Saint-Jacques, est liée à des changements de diversité spécifique au sein des communautés de Pseudo-nitzschia. Cette thèse avait pour objectif de caractériser la diversité écophysiologique au sein du genre Pseudo-nitzschia afin de mieux comprendre les facteurs qui influencent les différences de toxicité et de phénologies des efflorescences des principales espèces de Pseudo-nitzschia de Baie de Seine. Différents paramètres physiologiques, tels que la croissance, la production d’acide domoïque, les quotas élémentaires, les paramètres photosynthétiques, ainsi que les paramètres de cinétiques d’absorption du nitrate, du silicate et du phosphate, ont été étudiés chez quatre espèces : P. australis, P. pungens, P. fraudulenta et P. delicatissima. De plus, la variabilité intraspécifique a été évalué en étudiant de nombreuses souches par espèce et en estimant l’influence des variations de la taille cellulaire liées au cycle de vie de Pseudo-nitzschia sur les paramètres physiologiques. Les résultats montrent une importante diversité intraspécifique de la physiologie des quatre espèces, en partie liée à la taille cellulaire et parfois au cycle de vie. La connaissance de cette diversité intraspécifique a permis de caractériser différents traits physiologiques pour chaque espèce. Les résultats apportent des éléments de compréhension sur les conditions physiologiques favorables à la production d’acide domoïque et le rôle des éléments nutritifs dans le déterminisme des efflorescences des espèces de Pseudo-nitzschia. / In the diatom Pseudo-nitzschia, some species produce a neurotoxin called domoic acid responsible for amnesic shellfish poisoning in humans following the consumption of contaminated molluscs. In the Bay of Seine (English Channel), the variability of bloom toxicity is linked to differences in the Pseudo-nitzschia species diversity. The aim of this thesis was to characterize the ecophysiological diversity within the genus Pseudo-nitzschia in order to better understand the physiological parameters influencing differences in toxicity and bloom phenology among the main Pseudo-nitzschia species of the Bay of Seine. Growth, domoic acid production, elemental intracellular quotas, photosynthetic parameters and kinetic parameters for nitrate, phosphate, and silicic acid uptake were studied in four Pseudo-nitzschia species: P. australis, P. pungens, P. fraudulenta et P. delicatissima. The intraspecific variability was characterized with a multi-strain approach and also considering the effect of cell size on the studied physiological parameters. The results highlight the importance of the intraspecific variability in the physiology of the four species, in part due to cell size reduction linked to Pseudo-nitzschia life cycle. Delineate the intraspecific diversity allowed to characterize different physiological traits for each species. This work brings new information on the physiological conditions favorable to domoic acid production and on the role of nutrients in controlling Pseudo-nitzschia species diversity during blooms.