Dose-response assessment for impaired memory from chronic exposure to domoic acid among native American consumers of razor clams.

Domoic acid (DA) is a marine neurotoxin that accumulates in filtering shellfish during harmful algal blooms. A health protection limit of 20 ppm DA in razor clams (RC) has been set based principally upon an episode of acute DA toxicity in humans that included Amnesic Shellfish Poisoning among survivors. The objective of this study was to determine the dose-response relationship between estimated DA exposure through RC consumption and memory loss in Washington state Native Americans from 2005 to 2015. Results from total learning recall (TLR) memory scores were compared before and after the highest DA exposures. A decrease in TLR was related to DA dose (p < 0.01) regardless whether the effect was assumed to be transient or lasting, and whether the dose was expressed as an average daily dose or an average dose per meal. Benchmark dose modeling identified BMDL10 values of 167 ng/kg-day and 2740 ng/kg-meal assuming a transient effect, and 196 ng/kg-day and 2980 ng/kg-meal assuming no recovery of function occurs. These DA dose thresholds for a measurable memory function reduction observed in this study of clam consumers are well below the safe acute dose underpinning the current regulatory DA limit of 20 ppm (ca. 60 µg/kg).

Long-term neurotoxic effects of domoic acid on primary dopaminergic neurons.

Domoic acid, an excitatory neurotoxin produced by certain algae, reaches the food chain through accumulation in some sea organisms. To investigate its long-term neurotoxicity on dopaminergic neurons, prepared primary mesencephalic cell cultures were exposed to different concentrations of domoic acid (0.1, 1, 10, 100 µM) on the 8th day in vitro (DIV) for 4 days. On the 12th DIV, culture media were collected for measurement of lactate dehydrogenase and cultured cells were subjected to immunohistochemistry against tyrosine hydroxylase, neuronal nuclear antigen and glial fibrillary acidic protein, and fluorescence staining using H2DCFDA, JC-1 and Hoechst 33342 dyes. Moreover, roles of AMPA/KA and NMDA receptors in domoic acid neurotoxicity were also investigated. Domoic acid significantly decreased the number of dopaminergic neurons and adversely affected their morphology, and slightly reduced the expression of neuronal nuclear antigen and glial fibrillary acidic protein. Co-treatment of cultures with domoic acid and the AMPA/KA or NMDA receptor antagonists NBQX and MK-801 rescued significant number of dopaminergic neurons. Domoic acid significantly decreased red:green fluorescence ratio of JC-1 and did not affect production of reactive oxygen species and apoptotic cell death. In conclusions, the present study reveals that long-term treatment of primary mesencephalic cell culture with domoic acid significantly destroyed dopaminergic neurons. This effect appears to be attributed to activation of AMPA/KA and NMDA receptors and mitochondrial damage.

Neurotoxic effects of domoic acid on dopaminergic neurons in primary mesencephalic cell culture.

INTRODUCTION: Domoic acid is a potent marine neurotoxin produced by certain species of the diatom genus Pseudonitzschia. To our knowledge, there are no studies that have investigated neurotoxic effects of domoic acid on dopaminergic neurons. Accordingly, the present study was carried out to investigate the potential neurotoxic effects of domoic acid on dopaminergic neurons in primary mesencephalic cell culture. MATERIAL AND METHODS: Cultures prepared from embryonic mouse mesencephala (total of 250 embryos) were treated with different concentrations of domoic acid (0.1, 1, 10, 100 µM) on the 10th DIV for 48 h. On the 12th DIV, culture media were used for measurement of lactate dehydrogenase and cultured cells were subjected to immunostaining for tyrosine hydroxylase, neuronal nuclear antigen and glial fibrillary acidic protein, and fluorescence staining using H2DCFDA, JC-1 and DAPI stains. Moreover, roles of AMPA/KA and NMDA receptors in domoic acid neurotoxicity were also investigated. RESULTS: Domoic acid significantly decreased the number of dopaminergic neurons, decreased the expression of neuronal nuclear antigen and slightly affected astrocyte populations, and increased the release of lactate dehydrogenase into the culture media. AMPA/KA receptor antagonist NBQX but not NMDA receptor antagonist MK-801 significantly inhibited the neurotoxic effect of domoic acid on dopaminergic neurons. H₂DCFDA, JC-1 and DAPI fluorescence staining, respectively, revealed that DomA slightly raised ROS production, and significantly decreased mitochondrial membrane potential and increased apoptotic cell death of cultured cells. CONCLUSION: The current study presents for the first time the neurotoxic effects of domoic acid on dopaminergic neurons and this effect appears to be attributed to activation of AMPA/KA receptors on dopaminergic neurons.

Possible Effect of Muscle-relaxant Anaesthetics on Invasion, Adhesion and Migration of Breast Cancer Cells.

BACKGROUND: Aggressive surgical removal of the primary tumour is the preferred treatment, but with tumour progression, some tumours cannot be completely removed surgically. Anaesthetics are administered to facilitate surgery. However, anaesthetics act as a potential factor in tumour recurrence or metastasis. MATERIALS AND METHODS: Normal breast cells and cancer breast cells were treated with different doses of muscle-relaxant anaesthetics. The effects on breast cancer cell invasion, adhesion and migration of these anaesthetics were then investigated using in vitro models. RESULTS: With increasing dose of rocuronium bromide and suxamethonium chloride CRS, the number of MCF-10A and MCF-7 cells, but not that of MDA-MB-231 cells, decreased. There was almost no difference in the number of cells when the three cell lines were treated with different doses of vecuronium bromide. The study also demonstrated that rocuronium bromide promoted the invasion, adhesion and growth of MDA-231 cells, while suxamethonium chloride CRS had no effect. Interestingly, vecuronium bromide did not affect the motility and invasion of breast cancer cells significantly. CONCLUSION: An understanding of the effect of anaesthetics and their impact on tumour metastasis is important, thus using an appropriate aesthetic strategy could improve long-term survival in some patients.

Perinatal Domoic Acid as a Neuroteratogen.

In mammals, the period shortly before and shortly after birth is a time of massive brain growth, plasticity and maturation. It is also a time when the developing brain is exquisitely sensitive to insult, often with long-lasting consequences. Many of society's most debilitating neurological diseases arise, at least in part, from trauma around the time of birth but go undetected until later in life. For the past 15 years, we have been studying the consequences of exposure to the AMPA/kainate agonist domoic acid (DOM) on brain development in the rat. Domoic acid is a naturally occurring excitotoxin that enters the food chain and is known to produce severe neurotoxicity in humans and other adult wildlife. Our work, and that of others, however, has demonstrated that DOM is also toxic to the perinatal brain and that toxicity occurs at doses much lower than those required in adults. This raises concern about the current regulatory limit for DOM contamination that is based on data in adult animals, but has also allowed creation of a novel model of neurological disease progression. Herein, we review briefly the toxicity of DOM in adults, including humans, and describe features of the developing nervous system relevant to enhanced risk. We then review the data on DOM as a prenatal neuroteratogen and describe in detail the work of our respective laboratories to characterize the long-term behavioural and neuropathological consequences of exposure to low-dose DOM in the newborn rat.

Diagnosing domoic acid toxicosis in the California sea lion (Zalophus californianus) using behavioral criteria: A novel approach.

Domoic acid toxicosis in the California sea lion (Zalophus californianus) is difficult to diagnose using presence of toxin alone because the duration of domoic acid presence in blood and urine is generally less than 48 hr following exposure. Because domoic acid toxicosis is often suggested by presentation of behavioral abnormalities, we asked whether assessment of behavior might be useful for diagnostic purposes. We developed an ethogram to categorize behavioral data collected via continuous focal animal sampling. In total, 169 subjects were observed at a rehabilitation center. Sea lions with domoic acid toxicosis displayed head weaving (P < 0.0001) and muscle fasciculations (P < 0.01) significantly more often than animals in a comparison group. Dragging hind flippers and swift scanning were observed exclusively in animals from the domoic acid toxicosis group. The data show that behavioral diagnostic criteria can be effective in the diagnosis of domoic acid toxicosis in the California sea lion.

Characterization of renal toxicity in mice administered the marine biotoxin domoic Acid.

Domoic acid (DA), an excitatory amino acid produced by diatoms belonging to the genus Pseudo-nitzschia, is a glutamate analog responsible for the neurologic condition referred to as amnesic shellfish poisoning. To date, the renal effects of DA have been underappreciated, although renal filtration is the primary route of systemic elimination and the kidney expresses ionotropic glutamate receptors. To characterize the renal effects of DA, we administered either a neurotoxic dose of DA or doses below the recognized limit of toxicity to adult Sv128/Black Swiss mice. DA preferentially accumulated in the kidney and elicited marked renal vascular and tubular damage consistent with acute tubular necrosis, apoptosis, and renal tubular cell desquamation, with toxic vacuolization and mitochondrial swelling as hallmarks of the cellular damage. Doses≥0.1 mg/kg DA elevated the renal injury biomarkers kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin, and doses≥0.005 mg/kg induced the early response genes c-fos and junb. Coadministration of DA with the broad spectrum excitatory amino acid antagonist kynurenic acid inhibited induction of c-fos, junb, and neutrophil gelatinase-associated lipocalin. These findings suggest that the kidney may be susceptible to excitotoxic agonists, and renal effects should be considered when examining glutamate receptor activation. Additionally, these results indicate that DA is a potent nephrotoxicant, and potential renal toxicity may require consideration when determining safe levels for human exposure.

Low dose domoic acid influences spontaneous behavior in adult rats.

Domoic acid (DA) is a potent marine neurotoxine present in seafood. Intoxication by DA causes gastrointestinal symptoms like vomiting and diarrhoea and also the so-called amnesic shellfish poisoning (inflicting memory impairment and seizures). Since exposure to non-convulsive doses is relevant to the human health, we investigated the effect of low dose DA administration in adult Wistar rats. Rats were administered with DA at the dose 1.0 mg/kg and their behavior was monitored for one hour in three sessions. The first session started immediately after DA administration. The second and third session started one and two weeks later. After the third session, the histochemical analysis of the hippocampi of the animals was conducted (Fluoro-Jade B, bis-benzimide). DA increased time spent by locomotion and distance travelled in the second half of the first session and this effect was pronounced during the second and third session. Exploratory rearing was decreased by DA administration in the first half of the first session. DA influenced the grooming in biphasic manner (decrease followed by an increase of time spent by grooming). This biphasic trend was observed even two weeks after the DA administration. Histochemistry of DA treated rats did not confirm the presence of apoptotic bodies, Fluoro-Jade B positive cells were not found neither in CA1 nor CA3 area of the hippocampi. Our study revealed that a low dose of DA affect short and long-term the spontaneous behavior of rats without inducing neuronal damage.

Influence of low-dose neonatal domoic acid on the spontaneous behavior of rats in early adulthood.

Consumption of seafood containing toxin domoic acid (DA) causes an alteration of glutamatergic signaling pathways and could lead to various signs of neurotoxicity in animals and humans. Neonatal treatment with domoic acid was suggested as valuable model of schizophrenia and epilepsy. We tested how repeated early postnatal DA administration influences the spontaneous behavior of rats in adulthood. Rats were injected with 30 microg DA/kg from postnatal day (PND) 10 until PND 14. Their behavior was observed in the open field test for one hour (Laboras, Metris) at PND 35, PND 42 and PND 112. We did not find any difference between DA treated rats and animals injected with equivalent volume of saline in both test sessions at PND 35 and PND 42. DA rats at PND 112 exhibited significantly higher vertical and horizontal exploratory activity (tested parameters: locomotion, distance travelled, average speed reached during test, grooming and rearing) between the 30th-40th min of the test session and habituated over 10 min later. We conclude that at least in the given experimental design, neonatal DA treatment results in alteration of the spontaneous behavior of rats in adulthood.

Proteomic Analysis of Plasma from California Sea Lions (Zalophus californianus) Reveals Apolipoprotein E as a Candidate Biomarker of Chronic Domoic Acid Toxicosis.

Domoic acid toxicosis (DAT) in California sea lions (Zalophus californianus) is caused by exposure to the marine biotoxin domoic acid and has been linked to massive stranding events and mortality. Diagnosis is based on clinical signs in addition to the presence of domoic acid in body fluids. Chronic DAT further is characterized by reoccurring seizures progressing to status epilepticus. Diagnosis of chronic DAT is often slow and problematic, and minimally invasive tests for DAT have been the focus of numerous recent biomarker studies. The goal of this study was to retrospectively profile plasma proteins in a population of sea lions with chronic DAT and those without DAT using two dimensional gel electrophoresis to discover whether individual, multiple, or combinations of protein and clinical data could be utilized to identify sea lions with DAT. Using a training set of 32 sea lion sera, 20 proteins and their isoforms were identified that were significantly different between the two groups (p<0.05). Interestingly, 11 apolipoprotein E (ApoE) charge forms were decreased in DAT samples, indicating that ApoE charge form distributions may be important in the progression of DAT. In order to develop a classifier of chronic DAT, an independent blinded test set of 20 sea lions, seven with chronic DAT, was used to validate models utilizing ApoE charge forms and eosinophil counts. The resulting support vector machine had high sensitivity (85.7% with 92.3% negative predictive value) and high specificity (92.3% with 85.7% positive predictive value). These results suggest that ApoE and eosinophil counts along with machine learning can perform as a robust and accurate tool to diagnose chronic DAT. Although this analysis is specifically focused on blood biomarkers and routine clinical data, the results demonstrate promise for future studies combining additional variables in multidimensional space to create robust classifiers.

Ursolic acid improves domoic acid-induced cognitive deficits in mice.

Our previous findings suggest that mitochondrial dysfunction is the mechanism underlying cognitive deficits induced by domoic acid (DA). Ursolic acid (UA), a natural triterpenoid compound, possesses many important biological functions. Evidence shows that UA can activate PI3K/Akt signaling and suppress Forkhead box protein O1 (FoxO1) activity. FoxO1 is an important regulator of mitochondrial function. Here we investigate whether FoxO1 is involved in the oxidative stress-induced mitochondrial dysfunction in DA-treated mice and whether UA inhibits DA-induced mitochondrial dysfunction and cognitive deficits through regulating the PI3K/Akt and FoxO1 signaling pathways. Our results showed that FoxO1 knockdown reversed the mitochondrial abnormalities and cognitive deficits induced by DA in mice through decreasing HO-1 expression. Mechanistically, FoxO1 activation was associated with oxidative stress-induced JNK activation and decrease of Akt phosphorylation. Moreover, UA attenuated the mitochondrial dysfunction and cognitive deficits through promoting Akt phosphorylation and FoxO1 nuclear exclusion in the hippocampus of DA-treated mice. LY294002, an inhibitor of PI3K/Akt signaling, significantly decreased Akt phosphorylation in the hippocampus of DA/UA mice, which weakened UA actions. These results suggest that UA could be recommended as a possible candidate for the prevention and therapy of cognitive deficits in excitotoxic brain disorders.

Evaluation of circulating eosinophil count and adrenal gland function in California sea lions naturally exposed to domoic acid.

OBJECTIVE: To determine the effect of natural exposure to domoic acid (DA) on eosinophil counts and adrenal gland function in California sea lions (Zalophus californianus). DESIGN: Cross-sectional prospective study. ANIMALS: 39 California sea lions. PROCEDURES: Adult female sea lions admitted to a rehabilitation hospital during 2009 were classified into 1 of 3 groups (acute DA toxicosis, chronic DA toxicosis, or no DA exposure) on the basis of clinical signs, DA concentration in urine or feces, and hippocampal morphology. Endoparasite burden, eosinophil count, and serum cortisol and plasma ACTH concentrations were determined for each sea lion. For a subset of 8 sea lions, fecal glucocorticoid concentration after IM administration of cosyntropin was determined. RESULTS: Sea lions exposed to DA (acute DA toxicosis, n = 11; chronic DA toxicosis, 19) had higher eosinophil counts and lower serum cortisol concentrations, compared with values for sea lions with no DA exposure (9). Eosinophil count was not associated with endoparasite burden. Serum cortisol concentration was associated with plasma ACTH concentrations in sea lions from the no DA exposure group but not in sea lions in the acute or chronic DA toxicosis groups. Following cosyntropin injection, fecal glucocorticoid concentrations increased in all sea lions evaluated except 1. CONCLUSIONS AND CLINICAL RELEVANCE: In adult sea lions, eosinophilia may be a cost-effective biomarker for DA exposure and may reflect alterations in hypothalamic, pituitary gland, or adrenal gland function. Domoic acid exposure may have subtle health effects on marine animals in addition to induction of neurologic signs.

Enhanced neurogenesis in organotypic cultures of rat hippocampus after transient subfield-selective excitotoxic insult induced by domoic acid.

New neurons are continuously generated in the hippocampus and may play an important role in many physiological and pathological conditions. Here we present evidence of cell proliferation and neurogenesis after a selective and transient excitotoxic injury to the hippocampal cornu ammonis 1 (CA1) area induced by low concentrations of domoic acid (DOM) in rat organotypic hippocampal slice cultures (OHSC). DOM is an excitatory amino acid analog to kainic acid that acts through glutamate receptors to elicit a rapid and potent excitotoxic response. Exposure of slice cultures to varying concentrations of DOM for 24 h induced dose-dependent neuronal toxicity that was independent of activation of classic apoptotic markers. Treatment with 2 µM DOM for 24 h caused a selective yet transient neurotoxic injury in the CA1 subfield of the hippocampus that appeared recovered after 7 days of incubation in a DOM-free medium and showed significant microgliosis but no sign of astrogliosis. The DOM insult (2 µM, 24 h) resulted in a significant upregulation of cell proliferation, as assessed by 5-bromo-2-deoxyuridine (BrdU) incorporation, and a concurrent increase of the neuronal precursor cell marker doublecortin (DCX) within the subgranular zone of the dentate gyrus and area CA1. Neurogenesis occurred primarily during the first week after termination of the DOM exposure. Our study shows that exposure of OHSC to concentrations of DOM below those required to induce permanent neurotoxicity can induce proliferation and differentiation of neural progenitor cells that may contribute to recovery from mild injury and to develop abnormal circuits relevant to disease.

Ischemic cardiomyopathy following seizure induction by domoic Acid.

Exposure to the excitotoxin domoic acid (DOM) has been shown to produce cardiac lesions in both clinical and animal studies. We have previously shown that DOM failed to directly affect cardiomyocyte viability and energetics, but the development of this cardiomyopathy has remained unexplained. The present study compared effects of high-level seizure induction obtained by intraperitoneal (2 mg/kg) or intrahippocampal (100 pmol) bolus administration of DOM on development of cardiac pathologies in a rat model. Assessment of cardiac pressure derivatives and coronary flow rates revealed a significant time-dependent decrease in combined left ventricular (LV) systolic and diastolic function at 1, 3, 7, and 14 days after intraperitoneal administration and at 7 and 14 days after intrahippocampal DOM administration. LV dysfunction was matched by a similar time-dependent decrease in mitochondrial respiratory control, associated with increased proton leakage, and in mitochondrial enzyme activities. Microscopic examination of the LV midplane revealed evidence of progressive multifocal ischemic damage within the subendocardial, septal, and papillary regions. Lesions ranged from reversible early damage (vacuolization) to hypercontracture and inflammatory necrosis progressing to fibrotic scarring. Plasma proinflammatory IL-1α, IL-1ß, and TNF-α cytokine levels were also increased from 3 days after seizure induction. The observed cardiomyopathies did not differ between intraperitoneal and intrahippocampal groups, providing strong evidence that cardiac damage after DOM exposure is a consequence of a seizure-evoked autonomic response.

Immunomodulatory effects upon in vitro exposure of California sea lion and southern sea otter peripheral blood leukocytes to domoic acid.

During red tide bloom events, the marine diatom Pseudo-nitzschia produces the toxin domoic acid (DA), which has been associated with stranding and mortality events involving California sea lions (Zalophus californianus) and southern sea otters (Enhydra lutris). In addition to these well-documented DA-induced neurotoxic events, there is increasing concern that DA may exert chronic effects, such as immunomodulation, which may potentially increase an individual's susceptibility to a number of opportunistic infections following nonlethal exposure. We investigated the effects of DA on innate (phagocytosis and respiratory burst) and adaptive (mitogen-induced lymphocyte proliferation) immune functions with the use of peripheral blood leukocytes collected from healthy California sea lions and southern sea otters upon in vitro exposure to 0 (unexposed control), 0.0001, 0.001, 0.01, 0.1, 1.0, 10, and 100 microM DA. Domoic acid did not significantly modulate phagocytosis or respiratory burst in either species. For California sea lions, DA significantly increased ConA-induced T-lymphocyte proliferation upon exposure to DA concentrations ranging from 0.0001 to 10 microM, resulting in a nonlinear dose-response curve. There was no effect on lymphocyte proliferation at the highest concentration of DA tested. No effects on lymphocyte proliferation were observed in southern sea otters. Importantly, the in vitro DA concentrations affecting T-cell proliferation were within or below the range of DA in serum measured in free-ranging California sea lions following natural exposure, suggesting a risk for immunomodulation in free-ranging animals. Understanding the risk for immunomodulation upon DA exposure will contribute in the health assessment and management of California sea lions and southern sea otters, as well as guide veterinarians and wildlife rehabilitators in caring for and treating afflicted animals.

Neonatal exposure to low-dose domoic acid lowers seizure threshold in adult rats.

Exposing Sprague-Dawley rat pups to very low, sub-convulsant doses of domoic acid (DOM) during perinatal development has been previously shown to result in seizure-like activity in adulthood similar to partial complex epilepsy in humans, and to produce cellular and molecular changes in the dentate gyrus and area CA-3 of the hippocampus. To further these investigations we recorded electroencephalographical and behavioural activity in DOM and control rats following a normally sub-convulsant dose (25 mg/kg) of pentylenetetrazol. During this exposure, 50% of DOM-treated rats experienced a Stage V (tonic-clonic) seizure (X(2)((1))=5.33, P=0.021), indicating a lowering of generalized seizure threshold in these animals. In a separate experiment we explored focal seizure (afterdischarge) threshold as well as seizure propagation rates in treated rats, using a 25 consecutive day standard amygdala kindling paradigm. We report that the afterdischarge threshold for DOM-treated rats was significantly lower than controls (F((1,27))=7.117, P=0.013). No difference between groups was found in seizure progression as measured by afterdischarge duration, latency to first Stage V seizure, or latency to reach a fully kindled state (defined as five consecutive Stage V seizures). Timm staining to assess mossy fibre sprouting (MFS) in the hippocampus revealed a significant MFS increase relative to sham at the ventral level in both left and right inner molecular layer of the dentate gyrus for all DOM-treated animals, as well as in the dorsal stratum oriens of CA3 contralateral to electrode placement, and these increases were further enhanced by the kindling procedure. We conclude that perinatal exposure to subconvulsive doses of DOM results in permanent changes in neuronal excitability in the adult rat, as demonstrated by a lowering of both generalized seizure and focal afterdischarge threshold, and produces increased MFS following kindling.

Subclinical effects of saxitoxin and domoic acid on aggressive behaviour and monoaminergic turnover in rainbow trout (Oncorhynchus mykiss).

The algal produced neurotoxins saxitoxin and domoic acid may have serious effects on marine life and can be responsible for the intoxication of for instance sea mammals, sea birds and fish. Given that farmed fish cannot escape algal blooms, they may be more susceptible to intoxication than wild stocks. In the present study, subclinical effects of saxitoxin and domoic on aggressive behaviour and monoaminergic systems in the brain of the rainbow trout (Oncorhynchus mykiss) were investigated. The resident-intruder test was used to measure aggression where only the resident fish were subjected to the toxins and analysed for monoamines and their metabolites. The resident-intruder test was carried out on two consecutive days. On day one basal aggression was measured in the four groups. On day two three of the groups were injected with subclinical doses of one of the following: saxitoxin (1.752 microg/kg bw), domoic (0.75 mg/kg bw) or 0.9% saline solution. This was performed 30 min prior to the aggression test. Handling stress and injection affected aggressive behaviour, cortisol and the serotonergic system in telencephalic brain regions. Cortisol levels were elevated in all of the injected groups when compared to the control group. An increase in serotonergic turnover was evident when all injected groups were pooled and compared to the control group. All together this suggests that the handling stress in connection with the injection was similar in all of the three injected groups. In contrast to both the undisturbed control group and the toxin-injected groups, the saline-injected group displayed a reduction in aggressive behaviour which was evident in increased attack latency. Furthermore the domoic injected group displayed more aggressive attacks towards their conspecifics than the saline-injected group. Consequently the two toxins appear to mask the stress induced alteration in aggressive behaviour. Monoamine levels and monoaminergic turnover could not be demonstrated to be directly affected by the two toxins at the given doses in the investigated brain regions (dorsal and ventral parts of telencephalon, optic tectum, locus coeruleus, raphe nucleus, molecular and granular layer of cerebellum). This could indicate that the toxins mediate aggressive behaviour either through other systems than the monoaminergic systems, such as neuroactive amino acids, or that the mediation occurs in other brain regions.

In vivo seizure induction and affinity studies of domoic acid and isodomoic acids-D, -E and -F.

Domoic acid and its isomers are produced via algal blooms and are found in high concentrations in shellfish. Here, we assessed the acute seizurogenic potencies of isomers-D, -E and -F and their binding affinities at heterogeneous populations of KA receptors from rat cerebrum. In addition, binding affinities of all six isomers (Iso-A through -F) were assessed at AMPA receptors. Radioligand displacement studies indicated that the seizurogenic potency of Iso-F (E-configuration) closely correlates with its affinities at both KA and AMPA receptors, whereas isomers-D (Z) and -E (E), which exhibit distinctly lower seizurogenic potencies, are quite weak displacers. Previously observed functional potencies for isomers-A, -B and -C (Sawant et al., 2008) correlated with AMPA receptor affinities observed here. Taken together, these findings call into question previous structure-activity rules. Significantly, in our hands, Iso-D was ten-fold less potent than Iso-F. To further explain observed links between structural conformation and functional potency, molecular modeling was employed. Modeling results closely matched the rank order of potency and binding data observed. We further assessed the efficacy of isomers-D, -E and -F as pharmacological preconditioning agents. Acute preconditioning with low-dose Iso-D, -E or -F, before high-dose DA failed to impart behavioural tolerance. This study has shed new light on structural conformations affecting non-NMDA ionotropic glutamate receptor binding and functional potency, and provides a foundation for future work in areas of AMPA and KA receptor modeling.

Selective reductions in subpopulations of GABAergic neurons in a developmental rat model of epilepsy.

In the rat, early postnatal development is a critical period for neuronal migration, differentiation and network formation, requiring appropriate and timely glutamate and gamma-aminobutyric acid (GABA) signaling. Insults that affect either of these systems may result in increased excitatory activity, potentially leading to changes in neuronal proliferation and/or connectivity. We have previously shown that postnatal administration of low doses of domoic acid (DOM) can produce many of the behavioral and morphological changes found in current animal models of temporal lobe epilepsy (TLE), as well as the human condition. Using immunohistochemical techniques, we sought to characterize alterations in specific hippocampal GABAergic subpopulations at various locations along the septo-temporal axis in the DOM model. Results show decreased levels of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) in the ventral hilus and region- and sex-specific reductions in parvalbumin (PV)-containing immunoreactivity, but no alterations in somatostatin (SST) expression. These regional and sex-dependent changes in specific subpopulations of GABAergic interneurons may contribute to seizure development in this slowly progressing developmental model of TLE, and highlight how even subtle intervention may alter the interplay between glutamate and GABA systems during critical developmental stages.