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Joseph J Pancrazio

Publications and source records attributed to Joseph J Pancrazio.

12 recordsLinked to original sources
PubMed2006

Detection of marine toxins, brevetoxin-3 and saxitoxin, in seawater using neuronal networks.

There is a need for assay systems that can detect known and unanticipated neurotoxins associated with harmful algal blooms. The present work describes our attempt to monitor the presence of brevetoxin-3 (PbTx-3) and saxitoxin (STX) in a seawater matrix using the neuronal network biosensor (NNB). The NNB relies on cultured mammalian neurons grown over microelectrode arrays, where the inherent bioelectrical activity of the network manifested as extracellular action potentials can be monitored noninvasively. Spinal cord neuronal networks were prepared from embryonic mice and the mean spike rate across the network was analyzed before and during exposure to the toxins. Extracellular action potentials from the network are highly sensitive not only to purified STX and PbTx-3, but also when in combination with matrixes such as natural seawater and algal growth medium. Detection limits for STX and PbTx-3, respectively, are 0.031 and 0.33 nM in recording buffer and 0.076 and 0.48 nM in the presence of 25-fold-diluted seawater. Our results demonstrated that neuronal networks could be used for analysis of Alexandrium fundyense (STX-producer) and Karenia brevis (PbTx-producer) algal samples lysed directly in the seawater-based growth medium and appropriately diluted with HEPES-buffered recording medium. The cultured network responded by changes in mean spike rate to the presence of STX-or PbTx-producing algae but not to the samples of two non-STX and non-PbTx isolates of the same algal genera. This work provides evidence that the NNB has the capacity to rapidly detect toxins associated with cells of toxic algal species or as dissolved forms present in seawater and hasthe potential for monitoring toxin levels during harmful algal blooms.

Animals↗
PubMed2004

Gene expression profiles in the rat central nervous system induced by JP-8 jet fuel vapor exposure.

Jet propulsion fuel-8 (JP-8) is the predominant fuel for military land vehicles and aircraft used in the US and NATO. Occupational exposure to jet fuel in military personnel has raised concern for the health risk associated with such exposure in the Department of Defense. Clinical studies of humans chronically exposed to jet fuel have suggested both neurotoxicity and neurobehavioral deficits. We utilized rat neurobiology U34 array to measure gene expression changes in whole brain tissue of rats exposed repeatedly to JP-8, under conditions that simulated possible occupational exposure (6 h/day for 91 days) to JP-8 vapor at 250, 500, and 1000 mg/m(3), respectively. Our studies revealed that the gene expression changes of exposure groups can be divided into two main categories according to their functions: (1). neurotransmitter signaling pathways; and (2). stress response. The implications of these gene expression changes are discussed.

Animals↗
PubMed2004

Measuring synchronization in neuronal networks for biosensor applications.

Cultures of neurons can be grown on microelectrode arrays (MEAs), so that their spike and burst activity can be monitored. These activity patterns are quite sensitive to changes in the environment, such as chemical exposure, and hence the cultures can be used as biosensors. One key issue in analyzing the data from neuronal networks is how to quantify the level of synchronization among different units, which represent different neurons in the network. In this paper, we propose a synchronization metric, based on the statistical distribution of unit-to-unit correlation coefficients. We show that this synchronization metric changes significantly when the networks are exposed to bicuculline, strychnine, or 2,3-dioxo-6-nitro-l,2,3,4-tetrahydrobenzoquinoxaline-7-sulphonamide (NBQX). For that reason, this metric can be used to characterize pharmacologically induced changes in a network, either for research or for biosensor applications.

Action Potentials↗
PubMed2004

Pharmacological effects of the marine toxins, brevetoxin and saxitoxin, on murine frontal cortex neuronal networks.

Brevetoxins and saxitoxins (STXs), which are produced by marine dinoflagellates, are very potent neurotoxins targeting separate sites of the alpha subunit of voltage-dependent sodium channels (VDSCs). An attractive approach for marine toxin detection relies on pharmacological modulation of VDSCs expressed in cells or tissues. While these function-based cellular assays exhibit the required sensitivity, they are typically slow and have limited potential use for field applications. Cultured neuronal networks grown on substrate integrated microelectrode arrays (MEAs) have emerged as a robust and sensitive approach for environmental threat detection. The present work describes the rapid effects of brevetoxin-2 (PbTx-2) and STX on embryonic murine frontal cortex neuronal networks on MEAs. Network recording parameters such as mean spike rate, burst rate, burst duration, number of spikes per burst and spike amplitude were analyzed before and after exposure to the toxins. STX produced fast and reversible inhibition of all electrophysiological parameters with IC(50)s ranging between 1.2 and 2.2nM. Although PbTx-2 also caused inhibition of most of the network electrophysiological parameters, it produced an increase in burst duration at lower concentrations (EC(50)=15+/-2 nM, n=4) followed by inhibition at higher ones (IC(50)=63+/-4 nM, n=4). Exposure of frontal cortex networks to PbTx-2 and STX also caused differential effects on spike amplitude. This work demonstrates that cultured neuronal networks not only could be used for pharmacological characterization of marine toxins but they also provide a tool with unique properties for their detection.

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PubMed2003

A portable microelectrode array recording system incorporating cultured neuronal networks for neurotoxin detection.

Cultured neuronal networks, which have the capacity to respond to a wide range of neuroactive compounds, have been suggested to be useful for both screening known analytes and unknown compounds for acute neuropharmacologic effects. Extracellular recording from cultured neuronal networks provides a means for extracting physiologically relevant activity, i.e. action potential firing, in a noninvasive manner conducive for long-term measurements. Previous work from our laboratory described prototype portable systems capable of high signal-to-noise extracellular recordings from cardiac myocytes. The present work describes a portable system tailored to monitoring neuronal extracellular potentials that readily incorporates standardized microelectrode arrays developed by and in use at the University of North Texas. This system utilizes low noise amplifier and filter boards, a two-stage thermal control system with integrated fluidics and a graphical user interface for data acquisition and control implemented on a personal computer. Wherever possible, off-the-shelf components have been utilized for system design and fabrication. During use with cultured neuronal networks, the system typically exhibits input referred noise levels of only 4-6 microVRMS, such that extracellular potentials exceeding 40 microV can be readily resolved. A flow rate of up to 1 ml/min was achieved while the cell recording chamber temperature was maintained within a range of 36-37 degrees C. To demonstrate the capability of this system to resolve small extracellular potentials, pharmacological experiments with cultured neuronal networks have been performed using ion channel blockers, tetrodotoxin and tityustoxin. The implications of the experiments for neurotoxin detection are discussed.

Action Potentials↗
PubMed2003

Acute neuropharmacologic action of chloroquine on cortical neurons in vitro.

Chloroquine, a common quinolone derivative used in the treatment of malaria, has been associated with neurologic side-effects including depression, psychosis and delirium. The neuropharmacologic effects of chloroquine were examined on cultured cortical neurons using microelectrode array (MEA) recording and the whole-cell patch clamp technique. Whole-cell patch clamp records under current-clamp mode also showed a chloroquine-induced depression of the firing rate of spontaneous action potentials by approximately 40%, consistent with the observations with the MEA recording, although no changes in either the baseline membrane potential or input resistance were observed. Voltage clamp recordings of spontaneous post-synaptic currents, recorded in the presence of tetrodotoxin, revealed no obvious changes in either the amplitude or rate of occurrence of inward currents with application of chloroquine at 10 microM, suggesting that the fundamental molecular mechanisms underlying spontaneous synaptic transmission may not be affected by acute application of the drug. In contrast, a concentration-dependent inhibition of whole-cell calcium current was observed in the presence of chloroquine. These acute neuropharmacologic changes were not accompanied by cytotoxic actions of the compound, even after exposure of up to 500 microM chloroquine for 7 h. These data suggest that chloroquine can depress in vitro neuronal activity, perhaps through inhibition of membrane calcium channels.

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PubMed2003

Biological threat detection via host gene expression profiling.

With the increased threat posed by biological weapons, detection techniques for biothreat pathogens are critically needed to monitor and assess the severity of the illness once exposure has occurred. Current approaches for detecting biological threats are either time-consuming or highly specific but provide little information regarding pathogenicity. Genotyping of pathogens by PCR provides a fast and definitive means for identifying pathogens, but reliance on pathogen genotypic endpoints has several limitations. Current progress in DNA microarrays technology provides an alternative way to address the issues faced by traditional detection systems through host gene expression profiles of peripheral blood cells. We discuss the advantages and critical issues facing the use of host gene expression profiling for biological threat detection.

Bioterrorism↗
PubMed2002

Toluene inhibits muscarinic receptor-mediated cytosolic Ca2+ responses in neural precursor cells.

Toluene is widely used as a component in industrial solvents and many toluene-containing products are abused via inhalation. While many studies have demonstrated its inhibitory effects on neuronal activity, the effects of toluene on receptor signaling in proliferating and differentiating neural precursor cells are presently unclear. Here, using digital video microscopy and Ca2+ imaging, we investigated the effects of acute exposure to toluene on the function of muscarinic acetylcholine receptors (mAChRs) expressed in neural precursor cells. The neural precursor cells were isolatedfrom embryonic day 13 (E13) rat cortex and expanded in serum-free medium containing basic fibroblast growth factor (bFGF). We found that the acetylcholine (ACh) analog carbachol (CCh) induced a dose-dependent increase in cytosolic Ca2+, which was blocked by the muscarinic receptor antagonist atropine in a reversible manner. Toluene was added to the perfusion medium and concentrations of toluene in the medium were determined by gas chromatographic analysis. Following imaging, the cells were fixed and processed for 5-bromo-2'-deoxyuridine (BrdU, cell proliferation marker) and beta-tubulin (TuJ1, neuronal marker) immunostaining. In the 5 day culture, most cells continued to divide (BrdU+), while afew cells differentiated into young neurons (TuJ1-). The CCh-induced Ca2+ elevations in proliferating (BrdU+TuJ1-) neural precursor cells were significantly reduced by acute exposure to 0.15 mM toluene and completely blocked by 10 mM toluene. Toluene's inhibition of muscarinic receptor-mediated Ca2+ signaling was rapid, reversible and dose-dependent with an IC50 value 0.5 mM. Since muscarinic receptors mediate cell proliferation and differentiation during neural precursor cell development, these results suggest that depression of muscarinic signaling may play a role in toluene's teratogenic effect on the developing nervous system.

Animals↗
PubMed2002

Potential applications of DNA microarrays in biodefense-related diagnostics.

Recent years have witnessed a logarithmic growth in the number of applications involving DNA microarrays. Extrapolation of their use for infectious diagnostics and biodefense-related diagnostics seems obvious. Nevertheless, the application of DNA microarrays to biodefense-related diagnostics will depend on solving a set of substantial, yet approachable, technical and logistical problems that encompass diverse topics from amplification efficiency to bioinformatics.

Bioterrorism↗
PubMedDate not supplied

Cultured neuronal networks as environmental biosensors.

Contamination of water by toxins, either intentionally or unintentionally, is a growing concern for both military and civilian agencies and thus there is a need for systems capable of monitoring a wide range of natural and industrial toxicants. The EILATox-Oregon Workshop held in September 2002 provided an opportunity to test the capabilities of a prototype neuronal network-based biosensor with unknown contaminants in water samples. The biosensor is a portable device capable of recording the action potential activity from a network of mammalian neurons grown on glass microelectrode arrays. Changes in the action potential fi ring rate across the network are monitored to determine exposure to toxicants. A series of three neuronal networks derived from mice was used to test seven unknown samples. Two of these unknowns later were revealed to be blanks, to which the neuronal networks did not respond. Of the five remaining unknowns, a significant change in network activity was detected for four of the compounds at concentrations below a lethal level for humans: mercuric chloride, sodium arsenite, phosdrin and chlordimeform. These compounds--two heavy metals, an organophosphate and an insecticide--demonstrate the breadth of detection possible with neuronal networks. The results generated at the workshop show the promise of the neuronal network biosensor as an environmental detector but there is still considerable effort needed to produce a device suitable for routine environmental threat monitoring.

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PubMedDate not supplied

Sensitivity of the neuronal network biosensor to environmental threats.

It is widely acknowledged that there is a critical need for broad-spectrum environmental threat detection. While cells/tissue-based biosensors have been discussed for many years as a means of meeting this critical need, these kinds of systems have met with logistic concerns, in particular with regard to stability. Our group has been working with cultured neuronal networks, which have the capacity to respond to a wide range of neuroactive compounds and are sufficiently robust to be shipped to end users. The basis of operation involves extracellular recording using thin-film microelectrode arrays where spontaneous bioelectrical activity, that is, spike firing, can be monitored in a noninvasive manner conducive for potentially long-term measurements. This work describes the current status of our efforts for the fabrication of a portable biosensor that incorporates cultured neuronal networks grown over standardized microelectrode arrays. Based on our protocol for aqueous phase sample introduction under constant flow conditions, minimal variation in mean spike rate is observed, consistent with temporal stability, such that changes of > 10% are readily distinguished. To demonstrate the capability of this system, changes are reported in mean spike rate and network synchronization resulting from exposure to different model environmental threats, cadmium and strychnine. The sensitivity of this assay approach and implications of the experimental findings for environmental threat detection are discussed.

Animals↗