Variation in brevetoxin and brevenal content among clonal cultures of Karenia brevis may influence bloom toxicity.

Karenia brevis, the major harmful algal (HA) species in the Gulf of Mexico, produces a suite of brevetoxins and brevenal, a nontoxic brevetoxin antagonist. K. brevis growth is reported to be optimum at oceanic conditions, yet blooms are most problematic in coastal waters. Differences in growth rate, total brevetoxin production, brevetoxin profiles and brevenal production were evaluated among eight K. brevis clones grown at salinities of 35 and 27, but otherwise identical conditions. All measured parameters varied significantly among clones and the individual responses to decreased salinity varied as well. At 27, growth rates of four clones increased (Wilson, TXB3, SP1 and SP2), but decreased in three others (TXB4, SP3 and NBK) as compared to 35. Total brevetoxin cellular concentration varied up to approximately ten-fold among clones. For most clones (5 of 8), no significant difference in total toxin production between salinity treatments was observed; however, there was a shift in brevetoxin profiles to a higher proportion of PbTx-1 vs. PbTx-2 (in 7 of 8 clones). Brevenal production decreased in the majority of the clones (6 of 8) when grown at a salinity of 27. Results suggest that K. brevis produces more PbTx-1 and less brevenal in lower salinity waters.

The inhibition of CHO-K1-BH4 cell proliferation and induction of chromosomal aberrations by brevetoxins in vitro.

Brevetoxins (PbTxs) are highly potent trans-syn polyether neurotoxins produced during blooms of several species of marine dinoflagellates, most notably Karenia brevis. These neurotoxins act on voltage-sensitive sodium channels prolonging the active state. During red tides, the commercial fishing and tourism industries experience millions of dollars of lost revenue. Human consumption of shellfish contaminated with PbTxs results in neurotoxic shellfish poisoning (NSP). Additionally, blooms of K. brevis are potentially responsible for adverse human health effects such as respiratory irritation and airway constriction in coastal residents. There is little information regarding the full range of potential toxic effects caused by PbTxs. Recent evidence suggests that PbTxs are genotoxic substances. The purpose of this study was to determine if PbTxs could induce chromosomal aberrations and inhibit cellular proliferation in CHO-K1-BH4 cells, and if so, could the damage be negated or reduced by the PbTx antagonist brevenal. Results from the chromosomal aberrations assay demonstrated that PbTxs are potent inducers of CHO-K1-BH4 chromosome damage. Results from the inhibition of cellular proliferation assays demonstrated that PbTxs inhibit the ability of CHO-K1-BH4 cells to proliferate, an effect which can be reduced with brevenal.

Florida Red Tide: Inhalation Toxicity of Karenia brevis Extract in Rats.

Brevetoxins are neurotoxins produced by the marine dinoflagellate Karenia brevis. Histopathologic examination of marine mammals dying following repeated exposure of brevetoxins during red tide events suggests that the respiratory tract, nervous, hematopoietic, and immune systems are potential targets for toxicity in repeatedly exposed individuals. The purpose of this experiment was to evaluate the effects of repeated inhalation of K. brevis extract on these potential target systems in rats. Male Sprague-Dawley rats were exposed four hours/day, five days/week for up to four weeks to target concentrations of 200 and 1000 µg/L K. brevis extract (approximately 50 and 200 µg/L brevetoxin-like compounds; positive neurotoxicity in a fish bioassay). Control rats were sham exposed to air. Immunohistochemical staining of pulmonary macrophages indicated deposition of brevetoxin-like compound within the lung. However, exposure resulted in no clinical signs of toxicity or behavioral changes. There were no adverse effects on hematology or serum chemistry. No histopathological changes were observed in the nose, lung, liver, kidneys, lymph nodes, spleen, or brain of exposed rats. Immune suppression was suggested by reduced responses of spleen cells in the IgM-specific antibody-forming plaque cell response assay and reduced responses of lymphocytes to mitogen stimulation in vitro. Differences between responses observed in rats in this study and those observed in manatees may be a function of dose or species differences in sensitivity.

Brevetoxin Depuration in Shellfish via Production of Non-toxic Metabolites: Consequences for Seafood Safety and the Environmental Fate of Biotoxins.

During blooms of the dinoflagellate Karenia brevis, filter-feeders such as oysters and clams bioaccumulate brevetoxins, often to levels that are toxic to humans. In controlled aquarium experiments, we exposed live oysters to bloom levels of toxic K. brevis, followed by 10 weeks of exposure to non-toxic microalgae. Oysters were harvested weekly and analyzed for brevetoxins and brevetoxin metabolites to quantify toxin bioaccumulation and depuration. All of the PbTx-2 concentrated by oysters was immediately converted to a mixture of polar metabolites that were then slowly eliminated from the oysters. However, 90% of measured PbTx-3 was eliminated within two weeks of toxic exposure but without apparent biotransformation. Extracts of oysters containing high levels of PbTx-3 were toxic to mice by intraperitoneal (IP) injection. Extracts of oysters harvested after PbTx-3 had been eliminated were non-toxic despite high concentrations of PbTx-2 metabolites. Oysters collected in Florida during and after a bloom of K. brevis contained polar metabolites of PbTx-2 as well as PbTx-3, but no PbTx-2. Again, PbTx-3 concentration was a good predictor of mouse toxicity. One hundred percent conversion of PbTx-2 to polar metabolites was also accomplished in vitro by spiking oyster or clam homogenate with PbTx-2, followed by a brief incubation at room temperature. These PbTx-2 metabolites did not kill mice, either orally or by intraperitoneal injection, even at concentrations 30 times greater than toxic PbTx-3 levels.

The effects of haloperidol and clozapine on extracellular GABA levels in the prefrontal cortex of the rat: an in vivo microdialysis study.

Recent electrophysiological and pharmacological data indicate that dopamine enhances the activity of interneurons in the prefrontal cortex (PFC) and induces the release of GABA from these cells. We used in vivo microdialysis to examine the effects of two dopamine receptor antagonists on GABA release in the prefrontal cortex of awake, freely moving rats. Depolarization accomplished by local perfusion of potassium chloride or veratradine markedly increased extracellular GABA levels in the PFC. In contrast, local perfusion of TTX reduced extracellular GABA levels in the PFC. These data indicate that extracellular GABA is derived in part from neurons, and that extracellular levels of the inhibitory amino acid are impulse dependent. The acute administration of haloperidol weakly but significantly decreased extracellular GABA levels in the PFC; no effect of haloperidol on striatal extracellular GABA levels was observed. Systemic administration of the atypical antipsychotic drug clozapine markedly reduced extracellular GABA levels in the PFC, but did not alter striatal GABA levels. Thus, release of GABA from interneurons in the PFC is inhibited by two antipsychotic drugs. These data may suggest that different D2-like dopamine receptors are localized to pyramidal and nonpyramidal neurons in the cortex.

Modulation of extracellular gamma-aminobutyric acid in the ventral pallidum using in vivo microdialysis.

Intracranial microdialysis was used to investigate the origin of extracellular gamma-aminobutyric acid (GABA) in the ventral pallidum. Changes in basal GABA levels in response to membrane depolarizers, ion-channel blockers, and receptor agonists were determined. Antagonism of Ca2+ fluxes with high Mg2+ in a Ca(2+)-free perfusion buffer decreased GABA levels by up to 30%. Inhibition of voltage-dependent Na+ channels by the addition of tetrodotoxin also significantly decreased basal extracellular GABA concentrations by up to 45%, and blockade of Ca2+ and Na+ channels with verapamil reduced extracellular GABA by as much as 30%. The addition of either the GABAA agonist, muscimol, or the GABAB agonist, baclofen, produced a 40% reduction in extracellular GABA. GABA release was stimulated by high K+ and the addition of veratridine to increase Na+ influx. High K(+)-induced release was predominantly Ca(2+)-dependent, whereas the effect of veratridine was potentiated in the absence of extracellular Ca2+. Both high K(+)- and veratridine-induced elevations in extracellular GABA were inhibited by baclofen, whereas only veratridine-induced release was antagonized by muscimol. These results demonstrate that at least 50% of basal extracellular GABA in the ventral pallidum is derived from Ca(2+)- or Na(+)-dependent mechanisms. They also suggest that Na(+)-dependent release of GABA via reversal of the uptake carrier can be shown in vivo.

Apomorphine decreases extracellular GABA in the ventral pallidum of rats with 6-OHDA lesions in the nucleus accumbens.

Inhibition of a tonically active gamma-aminobutyric acid (GABA) projection from the nucleus accumbens to the ventral pallidum (VP) is thought to mediate the locomotor response elicited by dopamine in the nucleus accumbens. To evaluate this hypothesis, dopamine was depleted in the nucleus accumbens using 6-hydroxydopamine which produced an upregulated locomotor response to systemic apomorphine (0.2 mg/kg, s.c.). Simultaneously, the level of extracellular GABA in the VP was monitored using microdialysis. Apomorphine injection produced an elevation in locomotor activity only in the lesioned rats. While apomorphine reduced extracellular GABA in both control and lesioned rats, the reduction had an earlier onset and was more consistent in lesioned animals. Although the onset of the decline in extracellular GABA in the VP of lesioned rats corresponded to the onset of apomorphine-induced motor activity, a significant reduction in GABA persisted for 180 min, while the behavior returned to control levels by 60 min after injection. These data support a possible role for dopamine receptor-mediated inhibition of accumbal GABA neurons projecting to the VP in the initiation of locomotor activity.

High sensitivity HPLC assay for GABA in brain dialysis studies.

The measurement of GABA in brain dialysis experiments involves precolumn derivatization of the amino acid with o-phthaldehyde (OPA) in the presence of a thiol and subsequent high-pressure liquid chromatography with electrochemical detection. A method is described which employs an internal standard and coulometric preoxidation (+ 0.2 V) to eliminate unconjugated OPA/thiol prior to final oxidation (+ 0.4 V) of the derivatized sample. This allows for more efficient separation, providing a retention time for GABA of 9-10 min, a detection sensitivity of 2 to 5 x 10(-14) mol/sample and chromatographic stability for at least 2 weeks of daily use.

GABAA receptors containing alpha 1 and beta 2 subunits are mainly localized on neurons in the ventral pallidum.

The gamma-aminobutyric acid (GABA) projection from the nucleus accumbens to the ventral pallidum (VP) is important in the regulation of locomotion. Thus, stimulation and inhibition of GABAA receptors in the VP can alter locomotor activity. To determine whether the GABAA receptors are located presynaptically on accumbens efferents to the VP or postsynaptically on neurons intrinsic to the VP two experiments were performed. In the first, quinolinic acid lesions of the nucleus accumbens did not alter [3H]muscimol binding in the VP, while lesions in the VP significantly reduced (60-80%) binding as measured by light microscopic receptor autoradiography. In the second experiment, in situ hybridization with oligonucleotide probes for mRNAs of the alpha 1 and beta 2 subunits of the GABAA receptor was examined in the nucleus accumbens and VP. No mRNA for either subunit was observed in the nucleus accumbens, although many positively labeled neurons were present within the VP. By contrast, a moderate to high density of cells in both the nucleus accumbens and VP contained mRNA for glutamic acid decarboxylase. These data argue that the majority of GABAA receptors in the VP are not located presynaptically on axonal terminals originating from neurons in the nucleus accumbens.

gamma-Aminobutyric acid and mu-opioid receptor localization and adaptation in the basal forebrain.

In conclusion, GABAA receptors containing the alpha 1 subunit are localized on postsynaptic neurons in the ventral pallidum, mainly in the dorsolateral compartment and on presynaptic terminals in the nucleus accumbens. mu-opioid receptors are localized on postsynaptic neurons in both the nucleus accumbens and ventral pallidum, and therefore may be regulating presynaptic release of enkephalin from the accumbens-pallidal projection. Discrete lesions in the dorsomedial core of the nucleus accumbens will upregulate GABAA receptors in the dorsolateral compartment of the nucleus accumbens in a fashion similar to the upregulation of GABAA receptors in the globus pallidus after striatal lesions. However, larger lesions of the lateral core projection to the dorsolateral compartment of the ventral pallidum do not upregulate the GABAA receptors, suggesting that the mechanisms for upregulation of GABAA receptors are specific to the dorsomedial core or a smaller lesion. The uniqueness of the compartments within the nucleus accumbens and the ventral pallidum are supported by these receptor and mRNA analyses.

Amphetamine lowers extracellular GABA concentration in the ventral pallidum.

In vivo dialysis was performed in the ventral pallidum of the conscious rat, and extracellular levels of gamma-aminobutyric acid (GABA) measured. Forty minutes following peripheral administration of amphetamine (2.0 mg/kg, s.c.) the extracellular concentration of GABA was significantly reduced, in parallel with a significant elevation in motor activity. These data indicate that a decline in GABA transmission in the ventral pallidum may be important in the initiation of amphetamine-induced motor activity.

Somatodendritic release of endogenous dopamine: in vivo dialysis in the A10 dopamine region.

Using in vivo dialysis in the A10 region of the anesthetized rat, somatodendritic release of endogenous dopamine was demonstrated. Although endogenous dopamine release from the A10 region was enhanced by amphetamine pretreatment in a dose-related manner, the amount of dopamine released was markedly less than the axonal release of dopamine measured simultaneously in the nucleus accumbens.