Manganese toxicity is targeting an early step in the dopamine signal transduction pathway that controls lateral cilia activity in the bivalve mollusc Crassostrea virginica.

Manganese is a neurotoxin causing manganism, a Parkinson-like clinical disorder. Manganese has been shown to interfere with dopaminergic neurotransmission, but the neurotoxic mechanism involved is not fully resolved. In the bivalve mollusc Crassostrea virginica also known as the eastern oyster, beating rates of lateral cilia of the gill are controlled by dopaminergic-serotonergic innervation originating from their cerebral and visceral ganglia. Terminal release of dopamine activates D2-like receptors on these gill cells inhibiting adenylyl cyclase and slowing cilia beating rates. In C. virginica, manganese treatment disrupts this dopaminergic innervation of the gill, preventing the normal cilio-inhibitory response of lateral cells to dopamine. In this study an adenylyl cyclase activator (forskolin) and two different inhibitors (MDL-12,330A and SQ 22,536) were used to determine if manganese had any effects on the adenylyl cyclase step of the dopamine D2 receptor signal transduction pathway. The results showed that neither the adenylyl cyclase activator nor the inhibitors were affected by manganese in the control of lateral ciliary activity. This suggests that in C. virginica the mechanism of manganese toxicity on the dopaminergic control of lateral ciliary activity is targeting an early step in the D2R signal transduction pathway, which may involve interference with D2 receptor activation or alternatively some other downstream signaling activity that does not affect adenylyl cyclase.

Innervation of Gill Lateral Cells in the Bivalve Mollusc Crassostrea virginica Affects Cellular Membrane Potential and Cilia Activity.

Gill lateral cells of Crassostrea virginica are innervated by the branchial nerve, which contains serotonergic and dopaminergic fibers that regulate cilia beating rate. Terminal release of serotonin or dopamine results in an increase or decrease, respectively, of cilia beating rate in lateral gill cells. In this study we used the voltage sensitive fluorescent probe DiBAC4(3) to quantify changes in gill lateral cell membrane potential in response to electrical stimulation of the branchial nerve or to applications of serotonin and dopamine, and correlate these changes to cilia beating rates. Application of serotonin to gill lateral cells caused prolonged membrane depolarization, similar to plateau potentials, while increasing cilia beating rate. Application of dopamine hyperpolarized the resting membrane while decreasing cilia beating rate. Low frequency (5 Hz) electrical stimulations of the branchial nerve, which cause terminal release of endogenous serotonin, or high frequency (20 Hz) stimulations, which cause terminal release of endogenous dopamine, had the same effects on gill lateral cell membrane potentials and cilia beating rate as the respective applications of serotonin or dopamine. The study shows that innervation of gill lateral cells by the branchial nerve affects membrane potential as well as cilia beating rate, and demonstrates a strong correlation between changes in membrane potential and regulation of cilia beating rate. The study furthers the understanding of serotonin and dopamine signaling in the innervation and regulation of gill cilia in bivalves. The study also shows that voltage sensitive fluorescent probes like DiBAC 4(3) can be successfully used as an alternative to microelectrodes to measure changes in membrane potential of ciliated gill cells and other small cells with fast moving cilia.

Presence of Octopamine and an Octopamine Receptor in Crassostrea virginica.

Octopamine is a biogenic amine first identified in octopus. It has been well studied in arthropods and a few gastropods, serving as a neurotransmitter and hormone. The presence of octopamine has rarely been reported in bivalves and has not been reported in Crassostrea virginica. We utilized HPLC to identify and measure octopamine in cerebral ganglia, visceral ganglia, gill, palps, mantle, heart and hemolymph of C. virginica. Endogenous octopamine levels increased when animals were treated with tyramine, an octopamine precursor. A preliminary study in our lab found that octopamine has a cardio-excitatory action on C. virginica heart. In the present study we also used immunoblotting techniques to identify an octopamine-like receptor (Pan TAAR, trace amine-associated receptor) in oyster heart. The study confirms the presence of octopamine in the nervous system, innervated organs and hemolymph of C. virginica and identifies the presence of an octopamine-like receptor in heart, strengthening the contention that octopamine is important in the physiology of C. virginica as it is in other invertebrates.

The Presence of Histamine and a Histamine Receptor in the Bivalve Mollusc, Crassostrea virginica.

Histamine, a biogenic amine, is a neurotransmitter in neurons and sensory receptors in invertebrates. Histamine has rarely been reported in bivalves. We used HPLC with pre-column derivatization using 2,3-naphthalenedicarboxaldehyde (NDA) as a fluorescent labeling agent to measure histamine in ganglia, and peripheral tissues of the oyster Crassostrea virginica. We also used Western Blot technique to look for the presence of a histamine receptor in the mantle rim. HPLC results found histamine present in ng amounts in both the cerebral and visceral ganglia, as well as the mantle rim and other peripheral tissues of C. virginica. The study confirms and quantifies histamine as an endogenous biogenic amine in C. virginica in the nervous system and innervated organs. Western Blot technique also identified a histamine H2-like receptor present in sensory tissue of the oyster's mantle rim.

The Ability of PAS, Acetylsalicylic Acid and Calcium Disodium EDTA to Protect Against the Toxic Effects of Manganese on Mitochondrial Respiration in Gill of Crassostrea virginica.

Manganese (Mn) is an essential metal that at excessive levels in brain causes Manganism, a condition similar to Parkinson's disease. Previously we showed that Mn had a neurotoxic effect on the dopaminergic, but not serotonergic, innervation of the lateral ciliated cells in the gill of the Eastern Oyster, Crassostrea virginica. While the mechanism of action of Mn toxicity is not completely understood, studies suggest that Mn toxicity may involve mitochondrial damage and resulting neural dysfunction in the brain's dopaminergic system. In this study we utilized micro-batch chambers and oxygen probes to measure oyster gill mitochondrial respiration in the presence of Mn and potential Mn blockers. The addition of Mn to respiring mitochondria caused a dose dependent decrease in mitochondrial O(2) consumption. Pretreating mitochondria with calcium disodium EDTA (caEDTA), p aminosalicylic acid (PAS) or acetylsalicylic acid (ASA) before Mn additions, provided full protection against the toxic effects of Mn. While mitochondrial pretreatment with any of the 3 drugs effectively blocked Mn toxicity, none of the drugs tested was able to reverse the decrease in mitochondrial O(2) consumption seen in Mn treated mitochondria. The study found that high levels of Mn had a toxic effect on gill mitochondrial O(2) consumption and that this effect could be blocked by the drugs caEDTA, PAS and ASA. C. virginica continues to be a good model with which to investigate the mechanism that underlies manganese neurotoxcity and in the pharmacological study of drugs to treat or prevent Manganism.

Identification of dopamine D2 receptors in gill of Crassostrea virginica.

The lateral epithelial cells of gill of Crassostrea virginica are innervated by dopamine and serotonin nerves that regulate the beating rate of their lateral cilia. Terminal release of dopamine slows down the beating rate of the cilia, while serotonin release increases the beating rate. Previously, we showed that the dopaminergic, but not the serotonergic, mechanism regulating the beating rate of the lateral cilia was disrupted by manganese treatments and that this disruption was occurring postsynaptically, at the level of the dopamine receptor or further downstream in the signal transduction pathway. In humans manganese toxicity causes Manganism, a neurological disorder with clinical symptoms similar to Parkinson s disease. In this study we utilized pharmacological agents and an immunohistofluorescence technique to characterize the dopamine receptor type present on the lateral ciliated cells of C. virginica gill. Agonists and antagonists to dopamine D1 or dopamine D2 receptors were applied to gill sections and beating rates of the lateral cilia were measured by stroboscopic microscopy. The D2 agonists and D2 antagonists were effective in mimicking or blocking, respectively, the inhibitory actions of dopamine on lateral cilia beating, while application of either D1 agonists or D1 antagonists had no significant effect. In other experiments we used an epilume fluorescence microscopic fitted with FITC filters to view gill sections treated with a primary antibody against D2 receptors and a FITC-linked secondary antibody. Control gill sections without primary antibody exposure were similarly treated and viewed. The D2 antibody treated sections showed bright fluorescent receptor-antibody complexes present at the lateral ciliated cells and other areas of gill, when compared to controls. The results of our immunofluorescence study identify the presence of D2-like receptors on the lateral ciliated cells of C. virginica gill and our pharmacological results indicate that D2-like receptors are the postsynaptic dopamine receptors involved in the cilio-inhibitory response of the lateral cilia. The results of this study, when combined with our previous work, further suggest that the mechanism of action that underlies the dopaminergic neurotoxicity of manganese in gill of C. virginica involves disruption of D2-like receptors. C. virginica continues to provide a simple yet good model with which to study the physiology of dopaminergic systems as well as the pharmacology of drugs affecting biogenic amines.

Effects of Potential Therapeutic Agents on Copper Accumulations in Gill of Crassostrea virginica.

Copper is an essential trace element for organisms, but when in excess, copper's redox potential enhances oxyradical formation and increases cellular oxidative stress. Copper is a major pollutant in Jamaica Bay and other aquatic areas. Bivalves are filter feeders that accumulate heavy metals and other pollutants from their environment. Previously it was determined that seed from the bivalve Crassostrea virginica, transplanted from an oyster farm to Jamaica Bay readily accumulated copper and other pollutants into their tissues. In the present study we utilized Atomic Absorption Spectrometry to measure the uptake of copper into C. virginica gill in the presence and absence of three potential copper -blocking agents: diltiazem, lanthanum, and p-aminosalicyclic acid. Diltiazem and lanthanum are known calcium-channel blockers and p-aminosalicylic acid is an anti-infammarory agent with possible metal chelating properties. We also used the DMAB-Rhodanine histochemistry staining technique to confirm that copper was entering gill cells. Our result showed that diltiazem and p-aminosalicyclic acid reduced copper accumulations in the gill, while lanthanum did not. DMAB-Rhodanine histochemistry showed enhanced cellular copper staining in copper-treated samples and further demonstrated that diltiazem was able to reduce copper uptake. The accumulation of copper into oyster gill and its potential toxic effects could be of physiological significance to the growth and long term health of oysters and other marine animals living in a copper polluted environment. Identifying agents that block cellular copper uptake will further the understanding of metal transport mechanisms and may be beneficial in the therapeutic treatment of copper toxicity in humans.

The Effects of Copper and Copper Blocking Agents on Gill Mitochondrial O(2) Utilization of Crassostrea virginica.

While essential in trace amounts, excess copper is toxic to cells and tissues. Copper is a major aquatic pollutant. Previously, our lab demonstrated that tissues of the bivalve mollusc Crassostrea virginica readily accumulated copper and other metals from their surrounding environment. In this study we showed that O(2) consumption in isolated gill mitochondria of C. virginica was impaired by in vitro copper additions and that copper's deleterious effects on mitochondria respiration could be blocked by the presence of the membrane channel blocker Diltiazam.

Effects of p-Aminosalicylic acid on the neurotoxicity of manganese on the dopaminergic innervation of the cilia of the lateral cells of the gill of the bivalve mollusc, Crassostrea virginica.

The lateral cilia of the gill of Crassostrea virginica are controlled by a dopaminergic-serotonergic innervation. Dopamine is the neurotransmitter causing cilio-inhibition. High levels of manganese are neurotoxic to people, causing Manganism, a Parkinson-like disease. Clinical interventions for Manganism have not been very successful. Recently, p-Aminosalicylic acid (PAS) was reported as an effective treatment of severe Manganism in humans; however, its mechanism of action is unknown. Previously, we reported that manganese treatments caused disruption of the dopaminergic innervation of gill of C. virginica. Here we compared the effects of manganese on gill innervation in the presence of PAS, EDTA or Acetylsalicylic acid (ASA), and examined whether co-treating animals with PAS could block the deleterious effects of manganese on the oyster's dopaminergic innervation of the gill. Beating rates of the lateral cilia of the gill were measured by stroboscopic microscopy. Pre-treating gill preparations with PAS or EDTA blocked the neurotoxic effects of manganese, while ASA did not. In other experiments, animals exposed to three day treatments with manganese produced a dose dependent impairment of the dopaminergic, cilio-inhibitory system, which was decreased by co-treatment with PAS. The study shows that PAS protects the animal against neurotoxic effects of manganese and the mechanism of action of PAS in alleviating Manganism is more likely related to its chelating abilities than its anti-inflammatory actions.

The neurotoxic effects of manganese on the dopaminergic innervation of the gill of the bivalve mollusc, Crassostrea virginica.

We examined effects of manganese on the nervous system and innervation of lateral cilia of Crassostrea virginica. While essential in trace amounts, tissue manganese accumulation is neurotoxic, inducing Manganism, a Parkinson's-like disease in humans. Lateral cilia of the gill of C. virginica are controlled by a reciprocal serotonergic-dopaminergic innervation from their ganglia. Oysters were incubated 3 days in the presence of up to 1 mM manganese, followed by superfusion of the cerebral ganglia, visceral ganglia or gill with dopamine or serotonin. Beating rates of cilia were measured by stroboscopic microscopy of isolated gill preparations or gill preparations with the ipsilateral cerebral and/or visceral ganglia attached. Acute manganese treatments impaired the dopaminergic, cilio-inhibitory system, while having no effect on the serotonergic, cilio-excitatory system, which is in agreement with the proposed mechanism of manganese toxicity in humans. Manganese treatments also decreased endogenous dopamine levels in the cerebral and visceral ganglia, and gills, but not serotonin levels. We demonstrated that manganese disrupts the animal's dopaminergic system, and also that this preparation can be used to investigate mechanisms that underlie manganese neurotoxicity. It also may serve as a model in pharmacological studies of drugs to treat or prevent Manganism and other dopaminergic cell disorders.

Effects of p-Aminosalicylic acid on the Neurotoxicity of Manganese and Levels of Dopamine and Serotonin in the Nervous System and Innervated Organs of Crassostrea virginica.

Manganese is a neurotoxin causing Manganism in individuals chronically exposed to elevated levels in their environment. Toxic manganese exposure causes mental and emotional disturbances, and a movement disorder similar to Idiopathic Parkinsons Disease. Manganese interferes with dopamine neurons involved in control of body movements. Recently, p-aminosalicylic acid (PAS) is being used to alleviate symptoms of Manganism, but its mechanism of action is unknown. The eastern oyster, Crassostrea virginica, possesses a dopaminergic innervation of its gill. Oysters exposed to manganese have reduced levels of dopamine in the cerebral ganglia, visceral ganglia and gill, but not of norepinephrine, octopamine or serotonin. Those results are consistent with reported mechanisms of action of manganese in human and mammalian systems. In this study we determined the effects of PAS treatments on dopamine and serotonin levels in oysters exposed to manganese. Adult C. virginica were exposed to 500 µM and 1 mM of manganese with and without 500 µM and 1 mM of PAS by removing one shell and maintaining the animals in individual containers of aerated artificial sea water at 18° C for 3 days. Control animals were similarly treated without manganese or PAS. Dopamine and serotonin levels were measured by HPLC with fluorescence detection. PAS protected the ganglia and gill against the effects of 500 µM manganese, but not against the 1 mM manganese treatments. Serotonin levels were not affected by the treatments. The study demonstrates PAS can protect against reductions in dopamine levels caused by neurotoxic manganese exposure, but is concentration dependent. These findings may provide insights into the actions of PAS in therapeutic treatments of Manganism.

Its Time to Step into Science at Medgar Evers College.

Over the next decade, demand and job opportunities in science and engineering (S&E) are expected to grow. With so many S&E "baby-boomers" retiring, questions arise as to whether America will be able to attract enough young people into Science, Technology, Engineering and Mathematics (STEM) to maintain a S&E workforce that keeps up with what is becoming a more globally technologically and scientifically advancing society. Furthermore, considering recent projections of a nation more racially and ethnically diverse by mid-century, will America's future STEM workforce reflect the diversity projected for our growing U.S. population? In 2006, the authors received an award from the National Science Foundation (NSF) to direct a new initiative titled "STEP into Science." Funded under the Science, Technology, Engineering and Mathematics Talent Expansion Program (STEP) of the NSF Division of Undergraduate Education, the main goal of the project is to increase the number of STEM majors that graduate with baccalaureate degree, specifically B.S. degrees in Biology or Environmental Science. The program has had great success implementing the use of "peer recruiters" to attract more high school, transfer, and non-science college students into STEM majors and places emphasis on the role of undergraduate research experiences as a successful strategy to increase the quality and retention of science majors through their baccalaureate degree. Since the inception of the program, total STEM enrollment has more than doubled and the number of majors actively engaged in research has risen 38% with a concurrent increase in student research presentations at scientific conference, and the number of students receiving external research internships and travel awards to attend national conferences. The number of STEM graduates (both A.S. and B.S.) has also increased and the program anticipates that these and future STEP into Science graduates will continue on to Masters and Doctoral programs in STEM and ultimately enter rewarding careers in the science enterprise.

The nervous system control of lateral ciliary activity of the gill of the bivalve mollusc, Crassostrea virginica.

Lateral cilia of the gill of Mytilus edulis are controlled by a reciprocal serotonergic-dopaminergic innervation from their ganglia. Other bivalves have been studied to lesser degrees and lateral cilia of most respond to serotonin and dopamine when applied directly to the gill indicating a possible neuro or endocrine mechanism. Lateral cilia in Crassostrea virginica are affected by serotonin and dopamine, but little work has been done regarding ganglionic control of their cilia. We examined the role of the cerebral and visceral ganglia in innervating the lateral ciliated cells of the gill epithelium of C. virginica. Ciliary beating rates were measured in preparations which had the ipsilateral cerebral or visceral ganglia attached. Superfusion of the cerebral or visceral ganglia with serotonin increased ciliary beating rates which was antagonized by pretreating with methysergide. Superfusion with dopamine decreased beating rates and was antagonized by ergonovine. This study demonstrates there is a reciprocal serotonergic-dopaminergic innervation of the lateral ciliated cells, similar to that of M. edulis, originating in the cerebral and visceral ganglia of the animal and this preparation is a useful model to study regulatory mechanisms of ciliary activity as well as the pharmacology of drugs affecting biogenic amines in nervous systems.

Bioaccumulation and Tissue Distribution of Arsenic, Cadmium, Copper and Zinc in Crassostrea virginica Grown at Two Different Depths in Jamaica Bay, New York.

Historically, Jamaica Bay was a site of extensive oyster beds and shellfish culture leases that supported a significant oyster fishery in the New York area. The industrial and urban expansion of the early 1900's led to over-harvesting and a deterioration in water and bay sediment quality that coincided with shellfish decline and the ultimate disappearance of oysters from the bay. Over the past 50 years, efforts to arrest and reverse the pollution problems of Jamaica Bay have been undertaken but the area still contains metals and other pollutants at levels higher than NYS Water Quality Standards. Previous we showed that Crassostrea virginica seed transplanted to the bay had excellent growth and survival despite the bay's pollution problems. In this study we measured the one-year bioaccumulation and tissue distribution of four metals in C. virginica seed that were transplanted to the bay at two different depths: one foot from the surface and one foot above the sediment. Tissues of C. virginica were dissected, dried and digested in nitric acid. Arsenic, cadmium, copper and zinc levels were measured using electrothermal vaporization with deuterium lamp background correction in an atomic absorption spectrophotometer fitted with a THGA graphite furnace. Metals were distributed in the various tissues in µg/g dry weight amounts, which correlate well with published values for whole oysters grown in other polluted areas. Metal distributions were not homogeneous throughout the animals and in most of the tissues tested, oysters grown near the surface accumulated more metal than those positioned near bay sediment.

Growth and Survival of the American Oyster Crassostrea virginica in Jamaica Bay, New York.

Jamaica Bay is a major inlet opening to the Atlantic Ocean. It was abundant with oysters until early 1900's. Over-harvesting, pressure from predators, parasitic invasion and declining water quality often are cited as causes. Despite actions to arrest and reverse the pollution, oysters are not reestablished. We are studying factors relating to the rehabitation of Crassostrea virginica in Jamaica Bay to determine if the water quality and environmental conditions are suitable for their survival. Oysters placed in Jamaica Bay grew well when housed in protective containers and growth was influenced by placement near the sediment as compared to the surface. Oysters placed 1 foot above the sediment grew larger that those suspended 1 foot below the surface. Water temperature, pH, turbidity, salinity, conductivity, chlorophyll-a and dissolved O2 were taken to compare water quality at each site. To study growth and survival in a more natural condition, oyster seed and adults were placed just off the bottom in unprotected containers and photographed. After 1 year they are growing and surviving well and there has been evidence of reproduction. Thus far there are no serious signs of predation by crabs or starfish. The study shows that Jamaica Bay water quality is suitable for oyster growth under the various conditions of our experiments.