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Domoic acid (DA), a neurotoxin, is produced by marine algae and has caused toxications worldwide in animals and humans. However, the toxicokinetics of DA have not been fully evaluated, and information is missing on the disposition of DA following oral exposures at doses that are considered safe for human consumption. In this study, toxicokinetics of DA were investigated in cynomolgus monkeys following single doses of 5 µ g/kg DA intravenously, 0.075 mg/kg DA orally, and 0.15 mg/kg DA orally. After intravenous dosing, DA had a systemic clearance of 124 ± 71 (ml/h)/kg, volume of distribution at steady state of 131 ± 71 ml/kg and elimination half-life of 1.2 ± 1.1 hours. However, following oral dosing, the average terminal half-life of DA was 11.3 ± 2.4 hours, indicating that DA disposition follows flip-flop kinetics with slow, rate-limiting absorption. The absorption of DA was low after oral dosing with absolute bioavailability of 6% ± 4%. The renal clearance of DA was variable [21-152 (ml/h)/kg] with 42% ± 11% of the intravenous DA dose recovered in urine. A physiologically based pharmacokinetic model was developed for DA in monkeys and humans that replicated the flip-flop kinetics observed after oral administration and allowed simulation of urinary excretion and brain and kidney distribution of DA following intravenous and oral dosing. This study is the first to characterize DA disposition at exposure levels close to the current estimated tolerable daily intake and to mechanistically model DA disposition in a model species, providing important information of the toxicokinetics of DA for human safety assessment., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

The ubiquitous efflux transporter ABCC5 (ATP-binding cassette subfamily C member 5) is present at high levels in the blood-brain barrier, neurons, and glia, but its in vivo substrates and function are not known. Using untargeted metabolomic screens, we show that Abcc5(-/-) mice accumulate endogenous glutamate conjugates in several tissues, but brain in particular. The abundant neurotransmitter N-acetylaspartylglutamate was 2.4-fold higher in Abcc5(-/-) brain. The metabolites that accumulated in Abcc5(-/-) tissues were depleted in cultured cells that overexpressed human ABCC5. In a vesicular membrane transport assay, ABCC5 also transported exogenous glutamate analogs, like the classic excitotoxic neurotoxins kainic acid, domoic acid, and NMDA; the therapeutic glutamate analog ZJ43; and, as previously shown, the anti-cancer drug methotrexate. Glutamate conjugates and analogs are of physiological relevance because they can affect the function of glutamate, the principal excitatory neurotransmitter in the brain. After CO2 asphyxiation, several immediate early genes were expressed at lower levels in Abcc5(-/-) brains than in wild type brains, suggesting altered glutamate signaling. Our results show that ABCC5 is a general glutamate conjugate and analog transporter that affects the disposition of endogenous metabolites, toxins, and drugs., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (Copyright © 2014 by the American Society of Nephrology.)

Domoic acid (DA) causes neurological effects in multiple species upon exposure, including status epilepticus in pregnant sea lions and an epileptic disease state that commonly develops in juveniles. This study aims to define brain toxicokinetic parameters in the pregnant rat in the larger context of maternal-fetal toxin transfer. Specifically, Sprague-Dawley rats were exposed to a low observable effect level of 1.0 mg DA/kg intravenously at gestational day 20, and plasma, brain, and cerebrospinal fluid (CSF) samples were taken at discrete time points over 24 h. Domoic acid concentrations were determined by a tandem LC/MS method recently optimized for brain tissue and CSF. Data showed that 6.6% of plasma DA reached the brain, 5.3% reached the CSF, and DA levels were nearly identical in both brain and CSF for 12 h, remaining above the threshold to activate isolated hippocampal neurons for 2 h. The calculated terminal half-life of CSF was 4 h, consistent with the time for complete CSF regeneration, suggesting that CSF acts as a mechanism to clear DA from the brain.

Domoic acid (DA) is a potent neurotoxin that has both marine wildlife and human health impacts, including developmental effects during prenatal exposure in rodent models. However, little is known regarding DA toxicokinetics in the fetal unit during maternal-fetal transfer. Tissue distribution and toxicokinetics of DA were investigated in pregnant rats and their pups just prior to birth at gestational day 20. Pregnant Sprague Dawley rats were given an intravenous dose of 1.0 mg DA/kg and samples of maternal plasma, fetal plasma, placenta, amniotic fluid and fetal brain were taken at intervals over 24 h. Toxicokinetic parameters were determined using WinNonLin software analysis. Maternal plasma DA log concentration-time curves fit a two compartment pharmacokinetic profile, with alpha and beta half-lives of elimination of 26.9 and 297 min, respectively. Placenta had a C(max) of 752 ng/mL and a terminal half-life of 577 min. Maternal-fetal transfer between the plasma compartments was 31% with a fetal plasma C(max) of 86 ng/mL at 60 min and terminal half-life of 553 min. Amniotic fluid and fetal brain had overall averages of 27±12 ng/mL and 8.12 ng/g, respectively, and did not show evidence of elimination over 24 h. The longer fetal retention of DA, particularly in amniotic fluid, indicates that the fetus may be continually re-exposed during gestation, which could potentially lead to a disease state even at small exposure dose. This has implications for the California sea lions (Zalophus californianus), which exhibit an epilepsy-like disease that arises months after DA producing blooms., (Published by Elsevier Ireland Ltd.)

The western North Atlantic population of right whales (Eubalaena glacialis) is one of the most critically endangered of any whale population in the world. Among the factors considered to have potentially adverse effects on the health and reproduction of E. glacialis are biotoxins produced by certain microalgae responsible for causing harmful algal blooms. The worldwide incidence of these events has continued to increase dramatically over the past several decades and is expected to remain problematic under predicted climate change scenarios. Previous investigations have demonstrated that N. Atlantic right whales are being exposed to at least two classes of algal-produced environmental neurotoxins-paralytic shellfish toxins (PSTs) and domoic acid (DA). Our primary aims during this six-year study (2001-2006) were to assess whether the whales' exposure to these algal biotoxins occurred annually over multiple years, and to what extent individual whales were exposed repeatedly and/or concurrently to one or both toxin classes. Approximately 140 right whale fecal samples obtained across multiple habitats in the western N. Atlantic were analyzed for PSTs and DA. About 40% of these samples were attributed to individual whales in the North Atlantic Right Whale Catalog, permitting analysis of biotoxin exposure according to sex, age class, and reproductive status/history. Our findings demonstrate clearly that right whales are being exposed to both of these algal biotoxins on virtually an annual basis in multiple habitats for periods of up to six months (April through September), with similar exposure rates for females and males (PSTs: ∼70-80%; DA: ∼25-30%). Notably, only one of 14 lactating females sampled did not contain either PSTs or DA, suggesting the potential for maternal toxin transfer and possible effects on neonatal animals. Moreover, 22% of the fecal samples tested for PSTs and DA showed concurrent exposure to both neurotoxins, leading to questions of interactive effects. Targeted studies employing both in vivo and in vitro model systems represent the next logical step in assessing how and to what extent these algal biotoxins might compromise the health and reproduction of this endangered population., (Published by Elsevier Inc.)

The intestinal absorption mechanism of domoic acid (DA) was investigated using Caco-2 cells. DA is a tricarboxylic amino acid that contains a glutamic acid moiety, and causes deficits in short-term memory by binding to glutamate receptors as an agonist of glutamic acid. Caco-2 cell monolayers cultured on permeable membranes were incubated with 100 μM DA on either the apical or basolateral side, and the transcellular transport of DA was measured. The transcellular transport of DA from the apical to basolateral side was about twofold that in the opposite direction. The transcellular transport of DA from the apical side was optimal at a neutral pH, and was temperature- and Cl(-)-dependent, but was Na(+)-independent. Coincubation of DA with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an anion exchange inhibitor, significantly decreased the apical-to-basolateral transport of DA by 48%, and coincubation with probenecid (a non-specific anion transport inhibitor) significantly decreased the transport of DA by 31%. In contrast, coincubation with glutamic acid, succinic acid (a dicarboxylic acid), or citric acid (a tricarboxylic acid) did not decrease the transport of DA. These results suggest that the apical-to-basolateral transport of DA across the Caco-2 cell monolayers is mediated by DIDS-sensitive anion transporters., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

AMPA receptor-mediated responses to the agonist kainate differ from those of glutamate in two important respects. Glutamate is a full agonist that elicits strongly desensitizing responses, whereas kainate is a partial agonist with responses that are often described as weakly desensitizing or non-desensitizing. The efficacy of kainate relative to glutamate has previously been shown to be increased by mutations in the AMPA receptor ligand-binding cleft (Mano et al., 1996) and by coexpression with the auxiliary subunit stargazin (Tomita et al., 2005; Turetsky et al., 2005), but much less is known about factors that affect kainate desensitization. We therefore designed experiments to compare kainate and glutamate desensitization and efficacy in wild-type and mutant AMPA receptors expressed with and without stargazin in HEK293 cells. Desensitization to the two agonists was differentially affected by mutations in the helices participating in bonds between two subunits in the active state of the receptor (Sun et al., 2002), indicating that the protein interactions maintaining the stability of the dimer interface differ depending on which agonist is bound. Kainate efficacy was affected by factors distinct from ligand-binding cleft closure, including mutations in the dimer interface and channel vestibule as well as receptor composition. The increase in kainate responses for AMPA receptors coexpressed with stargazin was the result of both reduced kainate desensitization and increased kainate efficacy. These results provide critical new insights into the agonist dependence of both AMPA receptor activation and desensitization and the mechanism of the effects of stargazin on responses of partial agonists.

Ionotropic glutamate receptors principally mediate fast excitatory transmission in the brain. Among the three classes of ionotropic glutamate receptors, kainate receptors (KARs) have a unique brain distribution, which has been historically defined by (3)H-radiolabeled kainate binding. Compared with recombinant KARs expressed in heterologous cells, synaptic KARs exhibit characteristically slow rise-time and decay kinetics. However, the mechanisms responsible for these distinct KAR properties remain unclear. We found that both the high-affinity binding pattern in the mouse brain and the channel properties of native KARs are determined by the KAR auxiliary subunit Neto1. Through modulation of agonist binding affinity and off-kinetics of KARs, but not trafficking of KARs, Neto1 determined both the KAR high-affinity binding pattern and the distinctively slow kinetics of postsynaptic KARs. By regulating KAR excitatory postsynaptic current kinetics, Neto1 can control synaptic temporal summation, spike generation and fidelity.

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., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The neurotoxin domoic acid (DA), produced by diatoms Pseudo-nitzschia spp., is transferred to humans via consumption of contaminated bivalves. This study examines feeding mechanisms, namely reduced filtration, pre-ingestive rejection and poor absorption, that might explain the comparatively low DA levels commonly found in oysters during toxic Pseudo-nitzschia blooms. Clearance rate (CR), absorption efficiency (AE) of organic matter and selective rejection in pseudofeces of oysters (Crassostrea virginica) and mussels (Mytilus edulis) were investigated in relation to the DA levels accumulated during 2-wk, simultaneous exposure to toxic Pseudo-nitzschia multiseries. Effects of temperature and P. multiseries cell size were also tested to identify conditions, if any, under which oysters can accumulate unsafe DA levels. Oysters accumulated 3.0-7.5x less DA than mussels from a short-celled P. multiseries clone (length=24microm) at 12 degrees C. This was related to the 7.4-8.5x lower CRs determined for oysters relative to mussels at this temperature. Exposure to a longer-celled P. multiseries clone (81microm) resulted in up to 70x lower toxin levels in oysters compared to mussels, which was attributed to differential feeding selectivity. Mussels were unable to discriminate between long- and short-celled P. multiseries clones from a mixed suspension, whereas oysters were previously shown to preferentially reject long cells (>70microm) in pseudofeces. Both bivalves selectively rejected P. multiseries cells from mixed suspensions containing a flagellate but not another diatom. AE of organics from P. multiseries cells by oysters and mussels was comparably low (42 and 39%, respectively) and thus unlikely to explain their differential DA accumulation. CR and DA uptake by oysters were negligible at

Kainate receptor antagonists have potential as therapeutic agents in a number of neuropathologies. Synthetic modification of the convulsant marine toxin neodysiherbaine A (NDH) previously yielded molecules with a diverse set of pharmacological actions on kainate receptors. Here we characterize three new synthetic analogs of NDH that contain substituents at the C10 position in the pyran ring of the marine toxin. The analogs exhibited high-affinity binding to the GluK1 (GluR5) subunit and lower affinity binding to GluK2 (GluR6) and GluK3 (GluR7) subunits in radioligand displacement assays with recombinant kainate and AMPA receptors. As well, the natural toxin NDH exhibited approximately 100-fold selectivity for GluK2 over GluK3 subunits, which was attributable to the C8 hydroxyl group in NDH. We used molecular dynamic simulations to determine the specific interactions between NDH and residues within the ligand-binding domains of these two kainate receptor subunits that contribute to the divergent apparent affinities for the compound. These data demonstrate that interactions with the GluK1 subunit are preserved in analogs with substitutions at C10 in NDH and further reveal the determinants of selectivity and pharmacological activity of molecules acting on kainate receptor subunits, which could aid in design of additional compounds that target these receptors., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The king scallop Pecten maximus accumulates domoic acid, the main amnesic shellfish poisoning toxin, in the digestive gland for a long time. To try to find if the cause of this characteristic is the binding of the toxin to some cellular component, the subcellular distribution of domoic acid in the cells of the digestive gland was studied, by means of serial centrifugation, ultrafiltration and size exclusion chromatography (SEC). Domoic acid was found to be present mostly in soluble form in the cytosol, as more than 90% was found in the supernatant after a centrifugation of 1h at 45,000 x g, and passed a 10 kDa ultrafilter. The retention time of the peak with an absorption maximum of 242 nm--the one characteristic of domoic acid--observed in the SEC chromatograms of the scallop samples was found identical to be one of a reference solution of the toxin, indicating therefore, that domoic acid is free in the cytosol of the digestive gland of Pecten maximus. This finding turns the focus from binding to the lack of membrane transporters in this species of the scallop as the cause of the long retention time of domoic acid in this species., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Background: California sea lions have a large body burden of organochlorine pesticides, and over the last decade they have also been subject to domoic acid poisoning. Domoic acid poisoning, previously recognized in adult animals, is now viewed as a major cause of prenatal mortality. The appearance of a chronic juvenile domoic acid disease in the sea lions, characterized by behavioral abnormalities and epilepsy, is consistent with early life poisoning and may be potentiated by organochlorine burden., Objective: We investigated the interactive effect of DDT (dichlorodiphenyltrichloroethane) on neurodevelopment using a zebrafish (Danio rerio) model for seizure behavior to examine the susceptibility to domoic acid-induced seizures after completion of neurodevelopment., Methods: Embryos were exposed (6-30 hr postfertilization) to either o,p'-DDT or p,p'-DDE (dichlorodiphenyldichloroethylene) during neurodevelopment via a 0.1% dimethyl sulfoxide solution. These larval (7 days postfertilization) fish were then exposed to either the seizure-inducing drug pentylenetetrazol (PTZ) or domoic acid; resulting seizure behavior was monitored and analyzed for changes using cameras and behavioral tracking software., Results: Embryonic exposure to DDTs enhanced PTZ seizures and caused distinct and increased seizure behaviors to domoic acid, most notably a type of head-shaking behavior., Conclusion: These studies demonstrate that embryonic exposure to DDTs leads to asymptomatic animals at completion of neurodevelopment with greater sensitivity to domoic acid-induced seizures. The body burden levels of p,p'-DDE are close to the range recently found in fetal California sea lions and suggest a potential interactive effect of p,p'-DDE embryonic poisoning and domoic acid toxicity.

Background and Objectives: Prenatal exposure to asymptomatic doses of domoic acid (DA) causes learning and memory deficits later in life; therefore, we sought to measure distribution of DA in maternal plasma and brain, prenatal brain, and amniotic fluid 1 hr after exposure, a time frame that normally encompasses acute seizure behavior., Methods: Pregnant rats were given a single intravenous dose of DA (0.6 or 1.6 mg/kg body weight) at either gestational day (GD) 13 or GD20, which correspond to the beginning of rat embryo neurogenesis and the last day of gestation, respectively. Using a direct ELISA, dose-dependent levels of DA were detected in each sample matrix tested., Results: An average of 6.6 and 14 ng DA/g brain tissue was found in GD13 and GD20 prenatal rats, respectively. Brain concentrations of DA in the GD13 prenates were identical to amniotic fluid levels, consistent with no restriction for DA to enter the GD13 prenatal brain. At GD20 the prenatal brain contained half the concentration of DA in the amniotic fluid, and was approximately half that found in the brain of the dams. After 1 hr, fetal brain and amniotic fluid contained between 1 and 5% of DA found in the maternal circulation. The amniotic fluid levels of DA in this study were also within the same range measured in stranded California sea lions that showed reproductive failure., Conclusions: DA crosses the placenta, enters brain tissue of prenates, and accumulates in the amniotic fluid. Amniotic fluid appears to be a useful fluid to monitor DA exposure.

The solid-state high resolution magic angle spinning nuclear magnetic resonance (HRMAS NMR) technique was applied in this work to characterize and quantify the neurochemical changes in the rat hippocampus (CA1 or CA3) after local administration of kainic acid (KA). Intact tissue samples obtained from the KA treated and control brain samples were analyzed using HRMAS NMR. Metabolite profiles from NMR spectra of KA treated and control samples revealed the statistical significant decrease of N-acetylaspartate (NAA) and an increase of choline derivatives in the CA1 and CA3 directly receiving KA injection. Less extensive KA-induced metabolic changes were found in the hippocampi sample from the area contralateral to the site receiving KA administration. These results provided quantitative metabolic information on KA-induced neuronal loss and cell breakdown. In addition, the present study also revealed increased level of gamma-aminobutyric acid (GABA) and glutamate after KA treatment, suggesting that the cellular release of inhibitory and excitatory amino acids can be quantified using this method. KA induced microglia activation was evidenced by increased level of myo-insitol (myo-I). This study demonstrates that ex vivo HRMAS NMR is a useful tool for analyzing and quantifying changes of neurochemistry and cerebral metabolism in the intact brain.

Domoic acid (DA) is a potent neurotoxin naturally produced by some pennate diatom species of the genus Pseudo-nitzschia. It is well known that during harmful algal blooms fish can accumulate DA in the gastrointestinal (GI) tract and act as vectors of the toxin to higher trophic level piscivores, often with severe neurotoxic consequences to the predators. Although neurotoxicity and mass mortality have been observed in vertebrates (i.e. marine mammals and sea birds) feeding on contaminated fish, to date there has been no evidence of neurobehavioral toxicity in the fish vectors themselves. It has been hypothesized that fish may not absorb DA from the digestive tract, thus making them insensitive to dietary consumption of DA. To test this hypothesis, we performed oral gavage exposures followed by a time series of tissue dissections to characterize uptake, depuration, and tissue distribution of DA in fish. Intracoelomic (IC) injection exposures (which bypass the GI tract) were also performed to determine if coho neurons are neurologically susceptible to DA. Excitotoxic symptoms were observed in fish via IC injection at similar toxin levels that have been reported to induce excitotoxic symptoms in intraperitoneal (IP) exposures with mammalian models such as mice, suggesting that fish neurons have a similar sensitivity to DA as other vertebrates. Surprisingly, after oral gavage with ecologically relevant doses of DA, the toxin was detected in plasma collected from the dorsal aorta via a permanent intraarterial catheter within 15 min, yet excitotoxic symptoms were not observed. Additionally, DA was detected in liver, heart, spleen, kidney, muscle, brain and bile. These data indicate that although DA is absorbed from the gut, fish do not exhibit neuroexcitatory effects at maximum ecologically relevant oral doses of DA. Tissue distribution and DA uptake and depuration patterns suggest that a majority of the absorbed toxin is excreted via the kidneys and bile, thereby preventing toxic levels of DA from reaching sensitive nervous tissue. Additionally, greater than 20% of total IC administered DA doses were sequestered in bile within 1h of injection in five symptomatic fish, providing evidence for biliary sequestration of the toxin from blood. Here, we comprehensively describe the uptake, depuration, and tissue distribution patterns of DA and propose that renal and biliary processes may serve as primary routes of toxin clearance in fish.

Domoic acid (DA) is a highly toxic phycotoxin produced by bloom forming marine diatoms Pseudo-nitzschia spp. Bivalves can accumulate this toxin to a high level through their feeding activities, and thus illness or death in can occur in consumers of bivalves. In this study, king scallop, Pecten maximus, larvae were exposed to dissolved domoic acid (DA) for 25d, and the toxin accumulation and effects of harbouring this toxin were investigated. Scallop larvae incorporated DA continuously during the larval culture period and accumulated a maximum DA level of 5.21pgind(-1) when exposed to a solution of 50ngml(-1) dissolved DA. As a result of the DA treatment, larval growth, measured in terms of shell length and the appearance of the eye-spot, and larval survival were significantly compromised. This is the first study on DA incorporation dynamics in P. maximus larvae, signifying the potential of using shellfish larvae for the study on mechanisms of phycotoxin accumulation. The negative effect of DA exposure suggests that this toxin could possibly influence natural recruitment in P. maximus, and it may be necessary to protect hatchery-cultured scallop larvae from DA during toxic Pseudo-nitzschia blooms.

The effect of heat treatment on domoic acid (DA) content in soft tissues of mussels Mytilus edulis was investigated using high performance liquid chromatography. DA concentrations in whole flesh, hepatopancreas and tissue remainder were measured in fresh, steamed and autoclaved mussel flesh. Relative decreases in DA and tissue fluid following heat treatments of whole flesh were similar resulting in approximately equal concentrations of DA pre- and post-treatment. DA concentration decreased in the hepatopancreas and increased in tissue remainder suggesting some organ disruption of mussels during heat treatment. These findings suggest that heat treatments using either conventional steaming or autoclaving at 121 degrees C are not appropriate techniques to reduce DA in mussels during commercial processing. We also conclude that sample pre-treatment has a minimal effect on the result of a DA analysis on whole mussel tissues. The stability of DA at different temperatures within a shellfish matrix was separately tested. Reductions in DA concentration (ca. 3-7%) compensate for some of the discrepancies between what was found in the cooking fluids in the initial study and what was expected based on the whole flesh concentration of the uncooked material.

Using quantitative autoradiography, the present study examined ionotropic glutamatergic receptor binding sites using [3H]dizocilpine, [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate, and [3H]kainate in the posterior cingulate cortex of schizophrenia patients and matched controls. We found a significant increase in [3H]dizocilpine binding in the superficial layers (41%, p<0.001) and deep layers (30%, p=0.004) of the posterior cingulate cortex in the schizophrenia group compared with controls. No significant differences were observed in [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and [3H]kainate binding. In summary, the present study has for the first time demonstrated that the glutamatergic system is affected in the posterior cingulate cortex in schizophrenia patients. The fact that only the N-methyl-D-aspartate receptor densities are significantly altered suggests that this is unlikely to be caused by a simple decrease in glutamatergic transmission.

Domoic acid (DA) is a phycotoxin produced by some diatoms, mainly from the Pseudo-nitzschia genus, and has been detected throughout the marine food web. Although DA has been frequently found in cephalopod prey such as crustaceans and fish, little is known about DA accumulation in these molluscs. This study presents the first data showing relevant concentrations of DA detected in the common cuttlefish, Sepia officinalis, which is one of the most studied cephalopod species in the world. Domoic acid was consistently found throughout 2003 and 2004 in the digestive gland of cuttlefish reaching concentrations of 241.7 microg DA g(-1). The highest DA values were detected during spring and summer months, periods when Pseudo-nitzschia occur in the plankton. In fact, Pseudo-nitzschia blooms preceded the highest DA concentrations in cuttlefish. Evaluation of DA tissue distribution showed elevated DA concentrations in the digestive gland and branchial hearts. Further, DA isomers comprised a relevant percentage of the toxin profile, indicating degradation and biotransformation of the toxin in the branchial hearts. The common cuttlefish, like other cephalopod species, plays a central position in the food web and might be a new DA vector to top predators like marine mammals. Human intoxications are not expected since DA was only seldom detected in the mantle and even then in very low levels (max 0.7 microg DA g(-1)). However, in some countries whole juvenile animals are consumed (i.e. without evisceration) and in this case they might represent a risk to human health. This study contributes to understanding the occurrence of phycotoxins in cephalopods and reveals a new member of the marine food web able to accumulate DA.

Post-mortem studies have yet to produce consistent findings on cortical glutamatergic markers in schizophrenia; therefore, it is not possible to fully understand the role of abnormal glutamatergic function in the pathology of the disorder. To better understand the changes in cortical glutamatergic markers in schizophrenia, we measured the binding of radioligands to the ionotropic glutamate receptors (N-methyl D-aspartate, [3H]CGP39653, [3H]MK-801), amino-3-hydroxy-5-methyl-4-isoxazole ([3H]AMPA), kainate ([3H]kainate), and the high-affinity glutamate uptake site ([3H]aspartate) using in situ radioligand binding with autoradiography and levels of mRNA for kainate receptors using in situ hybridization in the dorsolateral prefrontal cortex from 20 subjects with schizophrenia and 20 controls matched for age and sex. Levels of [3H]kainate binding were significantly decreased in cortical laminae I-II (p = 0.01), III-IV (p < 0.05), and V-VI (p < 0.01) from subjects with schizophrenia. By contrast, levels of [3H]MK-801, [3H]AMPA, [3H]aspartate, or [3H]CGP39653 binding did not differ between the diagnostic cohorts. Levels of mRNA for the GluR5 subunit were decreased overall (p < 0.05), with no changes in levels of mRNA for GluR6, GluR7, KA1, or KA2 in tissue from subjects with schizophrenia. These data indicate that the decreased number of kainate receptors in the dorsolateral prefrontal cortex in schizophrenia may result, in part, from reduced expression of the GluR5 receptor subunits.

Purpose: Glutamate receptor 6 is strongly implicated in human refractory epilepsy and in kainic acid (KA)-induced status epilepticus (SE). In vitro pharmacologic studies with newer antiepileptic drugs (AEDs) are increasingly indicating the role of glutamate receptor 5 (GluR5) in epilepsy. Glutamate receptor 7 (GluR7) has been the least investigated in the context of epilepsy. We studied the messenger ribonucleic acid (mRNA) and protein expression of GluR5, GluR6, and GluR7 in rat hippocampus 72 h, 90 days, and 180 days after KA-induced SE., Methods: SE was induced by injecting KA intraperitoneally (i.p.) into adult rats. The hippocampi were isolated 72 h, 90 days, and 180 days after SE. Reverse transcription-polymerase chain reaction (RT-PCR) was performed for mRNA expression. Western blots determined the protein expression., Results: A significant increase was noted in GluR5 expression in KA-treated animals compared with controls at 72 h and 180 days, with no significant difference at the intervening 90-day point. Protein levels for GluR5 increased at 72 h and remained elevated until 180 days. GluR7 mRNA showed a significant decrease at 90 days after seizures. Neither the mRNA expression nor the protein levels of GluR6 differed from controls at any of the times after SE., Conclusions: KA-induced SE leads to an upregulation of GluR5 mRNA and protein and a downregulation of GluR7 mRNA in rat hippocampus, with no change in GluR6 mRNA or protein expression.

Domoic acid (DA), produced by the diatom genus Pseudo-nitzschia, is a glutamate analog and a neurotoxin in humans. During diatom blooms, DA can contaminate filter-feeding organisms, such as shellfish, and can be transferred by ingestion to higher trophic levels. Several intoxication events involving both humans and various marine mammals have been attributed to DA. Affected organisms show neurological symptoms such as seizures, ataxia, headweaving, and stereotypic scratching, as well as prolonged deficits in memory and learning. Neonatal animals have been shown to be substantially more sensitive to DA than adults. However, it has not been demonstrated whether DA can be transferred to nursing young from DA-exposed mothers. This study demonstrates transfer of DA from spiked milk (0.3 and 1.0 mg/kg) to the plasma of nursing neonatal rats and an overall longer DA retention in milk than in plasma after 8 hr in exposed dams. DA was detectable in milk up to 24 hr after exposure (1.0 mg/kg) of the mothers, although the amount of DA transferred to milk after exposure was not sufficient to cause acute symptoms in neonates.

The glutamate receptor (GluR) agonist-binding site consists of amino acid residues in the extracellular S1 and S2 domains in the N-terminal and M3-M4 loop regions, respectively. In the present study, we sought to confirm that the conserved ligand-binding residues identified in the AMPA receptor S1S2 domains also participate in ligand binding of GluR6 kainate receptors. Amino acid substitutions were made in the GluR6 parent at R523, T690, and E738 to alter their potential interactions with ligand. Mutant receptors were expressed in human embryonic kidney 293 cells, confirmed by Western blot analysis, and tested by [3H]kainate binding and patch-clamp recording. Each of the binding site mutations was sufficient to reduce [3H]kainate binding to undetectable levels and eliminate functional responses to glutamate or kainate. As with our studies of other nonfunctional mutants (Fleck et al., 2003), immunocytochemical staining and cell-surface biotinylation studies showed that the mutant receptors were retained intracellularly and did not traffic to the cell surface. Endoglycosidase-H digests and colocalization with endoplasmic reticulum (ER) markers demonstrated that the mutant receptors are immaturely glycosylated and retained in the ER. Immunoprecipitation, native PAGE, and functional studies confirmed that the GluR6-binding site mutants are capable of multimeric assembly, indicating their retention in the ER does not result from a gross protein folding error. Together, these results confirm the role of R523, T690, and E738 directly in ligand binding to GluR6 and further support our previous report that nonfunctional GluRs are retained intracellularly by a functional checkpoint in ER quality control.

We report the crystal structure of the glycosylated ligand-binding (S1S2) domain of the kainate receptor subunit GluR6, in complex with the agonist domoate. The structure shows the expected overall homology with AMPA and NMDA receptor subunit structures but reveals an unexpected binding mode for the side chain of domoate, in which contact is made to the larger lobe only (lobe I). In common with the AMPA receptor subunit GluR2, the GluR6 S1S2 domain associates as a dimer, with many of the interdimer contacts being conserved. Subtle differences in these contacts provide a structural explanation for why GluR2 L483Y and GluR3 L507Y are nondesensitizing, but GluR6, which has a tyrosine at that site, is not. The structure incorporates native glycosylation, which has not previously been described for ionotropic glutamate receptors. The position of the sugars near the subunit interface rules out their direct involvement in subunit association but leaves open the possibility of indirect modulation. Finally, we observed several tetrameric assemblies that satisfy topological constraints with respect to connection to the receptor pore, and which are therefore candidates for the native quaternary structure.

Loreclezole (5 mg/kg) exerted a significant protective action in amygdala-kindled rats, reducing both seizure and afterdischarge durations. The combinations of loreclezole (2.5 mg/kg) with valproate, clonazepam, or carbamazepine (applied at their subprotective doses) also exhibited antiseizure effect in this test. However, only two first combinations occurred to be of pharmacodynamic nature. Among several chemoconvulsants, bicuculline, N-methyl-D-aspartic acid and BAY k-8644 (the opener of L-type calcium channels) reversed the protective activity of loreclezole alone and its combination with valproate. On the other hand, bicuculline, aminophylline and BAY k-8644 inhibited the anticonvulsive action of loreclezole combined with clonazepam. The results support the hypothesis that the protective activity of loreclezole and its combinations with other antiepileptics may involve potentiation of GABAergic neurotransmission and blockade of L-type of calcium channels.

Parameters of [3H]uridine binding to synaptic membranes isolated from rat brain cortex (K(D)=71+/-4 nM, B(max)=1.37+/-0.13 pmol/mg protein) were obtained. Pyrimidine and purine analogues displayed different rank order of potency in displacement of specifically bound [3H]uridine (uridine>5-F-uridine>5-Br-uridine approximately adenosine>>5-ethyl-uridine approximately suramin>theophylline) and in the inhibition of [14C]uridine uptake (adenosine>uridine>5-Br-uridine approximately 5-F-uridine approximately 5-ethyl-uridine) into purified cerebrocortical synaptosomes. Furthermore, the effective ligand concentration for the inhibition of [14C]uridine uptake was about two order of magnitude higher than that for the displacement of specifically bound [3H]uridine. Adenosine evoked the transmembrane Na(+) ion influx, whereas uridine the transmembrane Ca(2+) ion influx much more effectively. Also, uridine was shown to increase free intracellular Ca(2+) ion levels in hippocampal slices by measuring Calcium-Green fluorescence. Uridine analogues were found to be ineffective in displacing radioligands that were bound to various glutamate and adenosine-recognition and modulatory-binding sites, however, increased [35S]GTPgammaS binding to membranes isolated from the rat cerebral cortex. These findings provide evidence for a rather specific, G-protein-coupled site of excitatory action for uridine in the brain.

The depuration kinetics of the domoic acid of four body fractions (digestive gland, adductor muscle, gonad+kidney and gills+mantle) of the scallop Pecten maximus was studied over 295 days. The scallops, which had acquired the toxins during a Pseudo-nitzschia australis episode that took place the week before the beginning of the experiment, were maintained in tanks with running seawater. All the body fractions, except the adductor muscle, decreased their domoic acid burden throughout the experiment. The amount of toxin in the muscle dropped sharply at the start of the experiment but increased again at the end, to levels that were higher than the initial ones. Several dynamic models of depuration kinetics, which included the depuration of each fraction (excluding the adductor muscle) and the transfers between them, were constructed, implemented and fitted to the data to obtain their parameters. The estimated depuration rates were very low, both considering and not considering the transfer of toxin between organs or the effect of weight loss. There were strong differences in the domoic acid burden of the body fractions studied but not between their depuration rates. No net transfer from the digestive gland, the tissue with highest domoic acid concentration, to the other fractions was found, as the inclusion of these processes in the models produced only a marginally better fit to the data. The depuration of domoic acid was slightly, but significantly, affected by biomass. Weight loss induced domoic acid loss, suggesting that part of the depuration may be produced by the direct loss of bivalve cells. The concentration or dilution effect, due to decreases or increases in biomass, documented for other species and toxins, has little importance in Pecten maximus.

The distribution of ionotropic glutamate receptors in transverse hippocampal sections and along the septotemporal hippocampal axis can be correlated to hippocampal connectivity, in particular to area- and layer-specific termination zones of afferents. However, in isolated organotypic hippocampal slice cultures developing without extrinsic afferent input no systematic studies exist about the distribution of glutamate receptors. In the present study we used receptor autoradiography to examine [3H]MK-801, [3H]AMPA and [3H]Kainate binding sites in hippocampal slice cultures prepared from 6-day-old rats. After 24 days in vitro layer-specific concentrations of [3H]MK-801, [3H]AMPA and [3H]Kainate binding sites were compared to age-matched hippocampi in situ (P30 rats). An obvious difference between hippocampal slice cultures and hippocampi in situ was a changed distribution of binding sites among the hippocampal areas showing a relative increase of [3H]MK-801 and [3H]AMPA binding sites in CA3 as compared to CA1 and to the dentate gyrus in the cultures. In CA1, however, the relative layer-specific distributions of [3H]MK-801 and [3H]AMPA binding sites were identical in hippocampal slice cultures and in hippocampi in situ. Interestingly, layer-specific binding of [3H]Kainate in the cultures exceeded that in the hippocampi in situ 3-5 times. Moreover, in the cultures the binding of the three ligands varied systematically showing gradients along the "superficiomembranal" axis. Cultures taken from different positions along the hippocampal axis differed with respect to concentrations of [3H]MK-801 and [3H]Kainate binding sites, but not of [3H]AMPA binding sites. The results suggest a massive sprouting and reorganisation of intrinsic projections in long-term hippocampal slice cultures.

Abnormalities of molecules associated with the glutamate synapse have been implicated in the pathophysiology of schizophrenia. Of the many glutamate receptors, those most commonly suggested to be involved in schizophrenia are the ionotropic subtypes, the NMDA, AMPA, and kainate receptors. Both the NMDA and AMPA subtypes have been extensively studied in postmortem brains of individuals with schizophrenia, but relatively little is known about the expression of the kainate subtype of glutamate receptor. In this study, we have determined cortical and striatal kainate receptor expression in brains from persons with schizophrenia and a comparison group, using both in situ hybridization and receptor autoradiography. At the level of subunit mRNA expression, a shift in subunit stoichiometry was evident in multiple regions of the prefrontal cortex, with increased expression of gluR7 mRNA and decreased expression of KA2 mRNA. Decreased kainate receptor binding was also observed in the subjects with schizophrenia, but was restricted to infragranular laminae of the prefrontal cortex. No differences in kainate receptor binding or subunit mRNA levels were found in striatum or occipital cortex, suggesting that these findings may be restricted to association cortex. These data add to the growing literature implicating ionotropic glutamate receptor disturbances in schizophrenia, and indicate that in addition to AMPA and NMDA receptors, the kainate receptors are also abnormally expressed in this illness.

Mature male and female mice from six inbred stains were tested for susceptibility to behavioral seizures induced by a single injection of cocaine. Cocaine was injected ip over a range of doses (50-100 mg/kg) and behavior was monitored for 20 minutes. Seizure end points included latency to forelimb or hindlimb clonus, latency to clonic running seizure and latency to jumping bouncing seizure. A range of strain specific sensitivities was documented with A/J and SJL mice being most sensitive and C57BL/6J most resistant. DBA/2J, BALB/cByJ and NZW/LacJ strains exhibited intermediate sensitivity. EEG recordings were made in SJL, A/J and C57BL/6J mice revealing a close correspondence between electrical activity and behavior. Additionally, levels of cocaine determined in hippocampus and cortex were not different between sensitive and resistant strains. Additional studies of these murine strains may be useful for investigating genetic influences on cocaine-induced seizures.

The interactions of S-nitrosoglutathione (GSNO) with the ionotropic glutamate receptors were studied on synaptic membranes isolated from the pig cerebral cortex. GSNO displaced the binding of [3H]glutamate, 3-[(R)-2-carboxypiperazin-4-yl] [3H]propyl-1-phosphonate ([3H]CPP), a competitive N-methyl-D-aspartate (NMDA) antagonist, and [3H]kainate, with IC50 values in the low micromolar range. It failed to displace (S)-5-fluoro-[3H]willardiine, a selective agonist of 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. Reduced and oxidized glutathione were almost as effective as GSNO in glutamate and CPP binding. Of the three, GSNO was the most potent in kainate binding. They all stimulated [3H]dizocilpine binding in a concentration-dependent manner. This effect was additive to that of glycine and not mimicked by NO donors such as S-nitroso-N-acetylpenicillamine, 5-amino-3-morpholinyl-1,2,3-oxadiazolium chloride (SIN-1) and nitroglycerin. We assume that GSNO may act as an endogenous ligand at the NMDA and non-NMDA classes of glutamate receptors. In this manner it may facilitate NO transfer and target its delivery to specific sites in these receptors.

Background: Intracerebroventricular (ICV) administration of kainic acid to rats produces limbic-cortical neuronal damage that has been compared to the neuropathology of schizophrenia., Methods: Groups of adult rats were administered ICV kainic acid and then assessed for neuronal loss and the expression of proteins relevant to mechanisms of neuronal damage after one and fourteen days. Neuronal loss was assessed by two-dimensional cell counting and protein expression was assessed by immunohistochemistry., Results: ICV kainic acid administration was associated with both immediate (day 1) and delayed (day 14) neuronal loss in the dorsal hippocampus. The immediate injury was largely limited to the CA3 hippocampal subfield, while the delayed injury included the CA1 subfield. Multiple mechanisms of cell death appeared to be involved in the delayed neuronal loss, as evidenced by changes in the expression of glutamate receptor subunits, heat shock protein and jun protein., Conclusions: ICV kainic acid administration to adult rats produces progressive damage to limbic-cortical neurons, involving both fast and slow mechanisms of cell death. Given the evidence for clinical deterioration, cognitive deficits and hippocampal neuropathy in some cases of schizophrenia, this animal model may be relevant for hypotheses regarding mechanisms of neurodegeneration in that disorder.

Domoic acid (DA) is an environmental neurotoxin to humans. This work examines whether repeated exposure to subsymptomatic or symptomatic nonlethal doses of domoic acid leads to enhanced symptomatic toxicity in ICR outbred and DBA inbred strains of laboratory mice. A multiple independent exposure paradigm was designed in which doses were administered intraperitoneally every other day for 7 days to achieve four separate exposures to domoic acid. We first examined the effect of repeated exposure on serum clearance of domoic acid. Serum domoic acid levels did not differ following a single or repeated exposure. We next examined the effect of repeated exposure on symptomatic toxicity. The mean toxicity scores did not show a significant difference between single and repeated exposures of either subsymptomatic (0.5 mg/kg) or symptomatic sublethal (2.0 mg/kg) doses of domoic acid. We then examined the effects of repeated domoic acid exposure on a second strain of mouse. DBA mice were chosen based upon their sensitivity to kainic acid-induced seizures; however, the ICR mice were more sensitive to low-dose domoic acid toxicity, particularly in terms of onset and duration of stereotypic scratching behavior. Our results indicate that both strains of mice have comparable concentration-dependent toxic responses to domoic acid; however, differences exist in the magnitude of the response and in specific symptoms. The mean toxicity scores did not show a significant difference when a single exposure (1.0 and 2.0 mg/kg domoic acid) and repeated exposure of the same dose were compared in the DBA mice. This study provides no evidence that short-term repeated exposure to domoic acid in laboratory mice alters domoic acid clearance from the serum, or leads to a more sensitive or a greater neurotoxic response.

Brain-stem afferents to the n. isthmo-opticus (NIO) and ectopic neurons (EN) of the centrifugal visual system (CVS) were determined in the pigeon using the retrograde transneuronal transport of the fluorescent dye Rhodamine beta-isothiocyanate (RITC) after its intraocular injection. In other experiments, either RITC was injected into various periocular tissues (controls) or the retrograde tracer Fluoro-gold (FG) was injected stereotaxically in the NIO. Following intraocular injections, the RITC retrograde labeling of cell bodies was observed contralaterally in the NIO and EN and transneuronally in layers 9/10 of the optic tectum, area ventralis-Tsai, zona peri-NIII, mesencephalic and pontine reticular formation (PRF), n. linearis caudalis-raphe, and bilaterally within a region referred to as zona peri-n.NVI (Zp-n.NVI) immediately underlying the abducens nerve nucleus. None of the above structures were labeled after RITC periocular injections. The FG labeling revealed that the tectal efferent neurons were mainly medium-sized, multipolar cells whose dendrites extended superficially to retino-recipient tectal layers 6 and 5. Quantitative measurements of the distribution of layers 9/10 RITC-labeled neurons indicated the highest densities to be localized within the ventral tectum corresponding to the representation of the dorsal retina and inferior visual field. We suggest that visual and nonvisual brain-stem afferents upon NIO and EN may play a role in the proposed mechanism of the avian CVS in attention, ground-feeding behavior, and modulation of retinal sensitivity.

The renal clearance (Clr) of the seafood toxin domoic acid (DA) was investigated in the rat. Following cannulation of the right femoral artery, the left femoral vein and the bladder of anaesthetized rats, a single bolus injection of either [3H]DA, [14C]p-aminohippuric acid (PAH) or [3H]inulin was administered through the venous cannula. Blood samples were taken from the arterial cannula at 1, 2, 10, 30, 50, 70, 90, 110 and 130 min following injection, and urine samples were collected at 20-min intervals starting from the time of bolus injection. Based on plasma concentration-time profiles, the total clearances (Clt) for DA, PAH and inulin were 9.12, 33.17 and 7.50 ml/min/kg body weight, respectively. The Clr calculated from urinary excretion rates were not significantly different from the Clt. Probenecid significantly reduced the Clr of PAH but did not affect that of DA. When DA was given at doses of 0.5 ng, 0.5 mg and 2.0 mg/kg body weight, the pharmacokinetic parameters Clt, Clr, elimination-rate constant and apparent volume of distribution at steady state were not statistically different between doses. The entire dose of 3H was recovered in the urine by 160 min after dosing, and analysis of urine samples by HPLC confirmed that the radiolabel (3H) was associated predominantly with the parent form of DA. The results of the present study demonstrate that DA is cleared from plasma primarily through the kidneys. DA clearance occurs primarily by renal glomerular filtration since its Clt is comparable with that of inulin, is less than that of PAH and is not affected by probenecid.

Molecular cloning of genes for the neurotrophin family and the identification of their high-affinity receptors have recently contributed to our understanding of neurotrophic interactions in the vertebrate nervous system. From their primary sites of synthesis, novel neuronal populations that may be sensitive to the neurotrophins have been identified. Protective roles for these factors following epileptic, ischemic, and hypoglycemic insults have been inferred. Documented neurotrophic actions on basal forebrain cholinergic neurons and mesencephalic dopaminergic neurons imply future clinical applications for the treatment of dementia of both Alzheimer's and Parkinson's disease. Studies on structure-function relationships of the neurotrophins hold promises for the development of specific receptor agonists and antagonists with possible basic science and clinical applications.

The autoradiographic distribution of binding sites for [3H]AMPA, [3H]kainate, [3H]MK-801 and [3H]L-glutamate and the in situ hybridization of GluR-A, GluR-B, GluR-C and GluR-D in rat spinal cord sections were determined. All of the radioligands had similar localisations with the dorsal horn being the most intensely labelled region. In the in situ hybridization experiments only the GluR-B mRNA was detected at high levels in the spinal cord with predominant expression in the substantia gelatinosa. These results support electrophysiological evidence for the presence of glutamate activated neurones in the spinal cord and suggest that these excitatory pathways are confined mainly to the dorsal horn.

Results are presented concerning the functional and morphological changes occurring after kainic acid injections into raphe dorsal nucleus in freely moving cats. The effects of kainic acid at two doses, 4 and 12 nmol, on electroencephalographic activity, behavior and cell morphology were examined on the day of injection, and on the 1st, 3rd, 6th, 15th, 30th and 65th day. A well-pronounced dose-dependent effect of kainic acid was established: in 50% of the animals injected with 4 nmol of kainic acid and in 85.7% of the animals injected with 12 nmol electroencephalographic and behavioral seizures appeared. The strength of the seizures in the animals receiving 12 nmol of kainic acid was different--from focal single epileptiform patterns first in raphe dorsal nucleus and then in other brain regions to generalized high-voltage ictal activity and in some animals to epileptic state. In all animals receiving 4 nmol of kainic acid and in 75% of the animals receiving 12 nmol of kainic acid, the electrical activity and behavior returned to normal several hours after or within two days following the kainic acid injection. The histopathological data obtained by light microscopy showed a dose-dependent cytoarchitectural disorganization (mainly chromatolytic changes) not only at the injection site but also in many other brain structures. The present results confirm our previous studies on cats in acute conditions showing enhancement of brain excitability level up to hyperexcitation (seizures) after kainic acid injection into the raphe dorsal nucleus.

The cause of the toxic mussel poisoning episode in 1987 was traced to a plankton-produced excitotoxin, domoic acid. Experiments were undertaken to quantitate the degree to which blood-borne domoic acid can permeate the microvasculature to enter the brain. Pentobarbital-anesthetized, adult rats received an i.v. injection of 3H-domoic acid which was permitted to circulate for 3-60 min. Transfer constants (Ki) describing blood-to-brain diffusion of tracer were calculated from analysis of the relationship between brain vs plasma radioactivity with time. Mean values (mL.g-1.s-1 X 10(6] for permeation into 7 brain regions (n = 10 rats) ranged from 1.60 +/- 0.13 (SE) to 1.86 +/- 0.33 (cortex, pons-medulla respectively), and carrier transport or regional selectivity in uptake were not evident. Nephrectomy prior to domoic acid injection resulted in the elevation of circulating plasma tracer level and brain uptake. The Ki values are comparable to those for other polar compounds such as sucrose, and indicate that the blood-brain barrier greatly limits the amount of toxin that enters the brain. Together with absorbed dosage, integrity of the cerebrovascular barrier and normal kidney function are important to the outcome of accidentally ingesting domoic acid.

Three questions are asked regarding the toxin kainic acid (KA). Does it destroy specific glial cells as well as neurons? Does KA gain access to the cytoplasm in intact cells and to which organelles does it bind? Intracerebral injections of tritiated KA into the pigeon (Columba livia) paleostriatal complex (basal ganglia) coupled with electron microscopic autoradiography revealed the following major points. Kainic acid destroyes oligodendrocytes, with pathophysiology apparent by 30 min after challenge with KA leading to cell destruction by 4 h. The response of astrocytes at the longest observation period (4 h) involves swelling of perivascular endfeet and processes in the neuropil. Reactive microglial-like cells show an accumulation of label in their cytoplasm, but no apparent morphological changes. The label appears in the cytoplasm of intact cells, both glia and neurons early after challenge with the toxin. Label is associated (bound) with mitochondria at an incidence significantly above chance at 30 min, 2 and 4 h after challenge with KA. Two hours after exposure to KA is the critical period where metabolic, physiological and morphological changes occur that lead to cell death. Cell destruction may be a consequence of KA-induced energy depletion. Kainate may interfere with adequate energy production by uncoupling glycolysis and the Krebs cycle in the mitochondria.

A grease-gap method for studying the pharmacology of CA1 hippocampal pyramidal cells was developed with use of rat hippocampal slices that included only area CA1 and the retrohippocampal area. These slices were transferred to a two-compartment superfusion chamber and the pyramidal cell bodies in area CA1 were separated from their axons in the subiculum with a grease barrier. The CA1 pyramidal cells were depolarized relative to their axons by superfusion with N-methyl-D-aspartate (NMDA), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate and L-glutamate. NMDA was unusually potent in the CA1-subiculum slice compared to other preparations. The NMDA receptor antagonists D(-)-2-amino-5-phosphonovalerate (D-AP5), phencyclidine and Mg2+ shifted the NMDA dose-response curve to the right in a parallel manner. Similarly, the quisqualate receptor antagonist pentobarbitone shifted the AMPA dose-response curve to the right. Schild plots for these antagonists had slopes insignificantly different from 1. These results are consistent with the presence of a substantial NMDA receptor reserve on CA1 pyramidal cells. They are also in line with the high density of excitatory amino acid receptors on CA1 hippocampal pyramidal cells and with the known pharmacological properties of these receptors. Grease-gap studies on the CA1-subiculum slice fill the need for a means of obtaining quantitative pharmacological data on CA1 pyramidal cells.

The effects of infusions of kainic acid (KA) into the paraventricular nucleus of the hypothalamus (PVH) on blood pressure (BP), heart rate (HR), baroreflex sensitivity, renal hemodynamics and urinary electrolyte (Na+, K+) excretion were examined in conscious rats. Provocative doses of KA (0.3, 1 and 3 ng/microliters.hr) were infused into the PVH to selectively stimulate neuronal cell bodies, but not fibers of passage. Unilateral PVH infusions of KA (1 ng/microliter.hr) produced excitatory behavior primarily consisting of occasional grooming and increased locomotor activity, and periodic increases in BP and HR. The mean slope of the baroreflex response (delta heart period/delta mean BP; phenylephrine infusion method) curves was decreased by 24% (P less than .01 vs. control) when measured 1 to 1.5 hr after initiation of PVH infusion. The extent of diffusion of [3H]KA (1 ng/microliter.hr) was examined 1 hr after PVH infusion. Radioactivity was detectable at levels above 3 x background 1.6, 2.0 and 3.5 mm, respectively, from the injection site in sagittal, coronal and horizontal planes, indicating fairly extensive distribution patterns. Paraventricular hypothalamic infusions of KA (3 ng/microliters.hr) produced more intense behavioral excitation with slightly greater increases in BP and HR as compared with 1 ng/microliter.hr infusions. Paraventricular hypothalamic infusions of KA (1 and 3 ng/microliters.hr) increased urine flow and urinary Na+ and K+ excretion without affecting urine osmolality, whereas infusions of KA (0.3 ng/microliters.hr) produced no alterations in behavior, BP, HR, renal hemodynamics or renal excretory function.(ABSTRACT TRUNCATED AT 250 WORDS)