Detection of Marine Toxins, Brevetoxin-3 and Saxitoxin, in Seawater

Dec 14, 2005 - The networks were perfused with either the toxin or algal cell solutions at a flow rate of 1 mL/min until a new stable baseline of mean...
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Environ. Sci. Technol. 2006, 40, 578-583

Detection of Marine Toxins, Brevetoxin-3 and Saxitoxin, in Seawater Using Neuronal Networks N A D E Z H D A V . K U L A G I N A , * ,† CHRISTINA M. MIKULSKI,‡ SAMUEL GRAY,† WU MA,† GREGORY J. DOUCETTE,‡ JOHN S. RAMSDELL,‡ AND J O S E P H J . P A N C R A Z I O †,§ Center for Bio/Molecular Science and Engineering, Code 6900, Naval Research Laboratory, Washington, D.C. 20375, and Marine Biotoxins Program, Center for Coastal Environmental Health and Biomolecular Research, National Ocean Service, National Oceanic and Atmospheric Administration, Charleston, South Carolina 29412

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 HEPESbuffered recording medium. The cultured network responded by changes in mean spike rate to the presence of STXor PbTx-producing algae but not to the samples of two nonSTX 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 has the potential for monitoring toxin levels during harmful algal blooms.

* Corresponding author phone: 202-767-0903; fax: 202-767-9594, e-mail: [email protected]. † Naval Research Laboratory. ‡ National Oceanic and Atmospheric Administration. § Current address: National Institutes of Health, NINDS, Bethesda, MD 20892. 578

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Introduction Harmful algal blooms (HABs) that produce very potent neurotoxins such as brevetoxins (PbTxs) and saxitoxins (STXs) have increased in frequency, intensity, and geographical distribution over the past decade (1). HABs harm many industries relying upon fishing and agriculture. Exposure to HAB toxins, which could result from ingestion of contaminated seafood, could lead to the potentially fatal paralytic shellfish poisoning in the case of saxitoxins or neurotoxic shellfish poisoning in the case of brevetoxins (2-3). Exposure to brevetoxin aerosols is common and leads to respiratory distress (4). Structure- and function-based bioassays are the main approaches for detection of algal toxins. Structure-based methods, such as immunoassays, have been widely applied for the identification and quantification of known analytes; however, these methods cannot address the needs for detection of unanticipated threats. Function-based assays for detection vary greatly in their complexity, biological endpoints, and practicality for continuous monitoring in field applications. Among the function-based methods, both mouse bioassays and in vitro measures have been used. Although whole-animal assays are frequently the accepted method for determining toxicity levels, major limitations of this approach include the requirement of a large number of animals, difficulties in quantification (5), and an emerging resistance to live animal testing in many countries. Various in vitro methods have been proposed for detecting STX and PbTx (6-9). At the core of these in vitro methods is the toxininduced modulation of voltage-gated sodium channel (VGSC) activity (10-14). We demonstrated recently that cultured frontal cortex neuronal networks grown over microelectrode arrays (MEAs) are very sensitive to purified STX and PbTx-2 at nanomolar concentrations (15). Moreover, the toxins produced specific effects on electrophysiological parameters, such as burst duration and spike amplitude. However, given the fact that the ionic constituents and concentrations in seawater differ greatly from those in neuronal cell culture media, a major question emerged as to whether this approach is sufficiently robust for the detection of algal toxins in the presence of natural seawater, a milieu that mimics environmental field samples. Considering the complexity of constituents of seawater field samples, it is possible that the sensitivity of the assay to the marine toxins could be altered. To address this question, we examined the sensitivity of cultured neuronal networks to STX and PbTx-3 in complex matrixes including both algal culture medium and natural seawater. In this paper, we demonstrate that the neuronal networks are sufficiently robust to tolerate administration of diluted seawater samples in neuronal culture medium with little or no change in the nanomolar sensitivity to these marine algal toxins. Moreover, we show that sonicated samples of actively growing, toxin-producing algal cultures induce changes in neuronal network activity consistent with the toxin concentration as determined by a neuronal network biosensor (NNB). This work provides evidence that the NNB has the capacity to rapidly detect toxins associated with toxic algal species raising the possibility of its use in detection of HAB toxins directly from field samples.

Materials and Methods MEAs, Cell Culture, and Recording. Spinal cord neuronal networks were purchased from Applied Neuronal Network Dynamics, Inc. (ANND, Dallas, TX) and delivered to the U.S. 10.1021/es051272a CCC: $33.50

 2006 American Chemical Society Published on Web 12/14/2005

FIGURE 1. (A) Spinal cord neuronal network cultured over a microelectrode array (MEA) substrate. Microelectrode contacts are approximately 10 µm in diameter. (B) Representative extracellular recordings of action potentials or spikes on two individual channels of spinal cord culture. Naval Research Laboratory (NRL) by overnight commercial service. Network preparations have been previously described by Gross et al. (16). Briefly, cells were dissociated from spinal cord tissue of embryonic Hsd:ICRb mice and seeded on an MEA surface with 64 recording sites. Figure 1A shows a spinal cord neuronal network cultured over an MEA substrate. Microelectrode contacts are approximately 10 µm in diameter. MEAs with seeded cells were maintained at 37 °C in Dulbecco’s Essential Medium supplemented with 5% horse serum in 90% air and 10% CO2. The neuronal networks were stored in an incubator until use. It is important to note that neurons cultured over microelectrode arrays have shown regular electrophysiological behavior and stable pharmacological sensitivity for over 9 months (17, 18). In fact, their precise methodological approach generates a co-culture of glial support cells and randomly seeded neurons, resulting in spontaneous bioelectrical activity ranging from stochastic neuronal spiking to organized bursting and long-term oscillatory activity. All electrophysiological measurements were performed using the neuronal network biosensor (NNB) incorporated in a portable recording system that was developed in the NRL. The system is capable of maintaining NNB at 37 °C under controlled flow conditions (15, 19). To establish concentration-dependency, networks were perfused at a flow rate of 1 mL/min using a recirculating medium reservoir such that the total medium volume was 40 mL. The networks were perfused continuously with control medium for at least 30-40 min before any toxin introductions. Control recording solution contained minimum essential medium (MEM) supplemented with 25 mM glucose, 40 mM N-[2-hydroxyethyl]-piperazine-N-[2-ethanesulfonic acid] (HEPES), and 26 mM NaHCO3 at pH 7.4. A 2× solution of the reagents at pH 7.4 was also prepared for dilution of sterilized natural seawater (Vero Beach, Florida), and algal samples to produce a recording medium suitable for the cultured neuronal networks. Spike activity from cultured neuronal networks was quantified using mean spike rate. Typically, at least 5 channels were used to calculate mean spike rate. Data were sampled at a rate of 40 kHz per channel and detected using a thresholdcrossing algorithm with a threshold of 40 µV with 100 µs set as the minimum time above a threshold. Mean spike rate was calculated across all active channels in 1-min intervals following computation of the spike rate of each channel. For some of the results provided, the spike rate was normalized according to the following relationship: YN ) (Yt1/Y01 + Yt2/ Y02 + ... + Ytn/Y0n)/n, where n is the number of channels used in a particular study; YN is normalized mean spike rate; Ytn is spike rate of a single channel “n” in the presence of a toxin; and Y0n is a spike rate of control baseline of a single channel “n”.

Toxins. STX (diacetate) and PbTx-3 standards were obtained from Calbiochem (San Diego, CA) and Sigma-Aldrich (St. Louis, MO), respectively. STX intermediate stock solution (2.3 µM) was prepared in deionized water. PbTx-3 was dissolved in 1% ethanol at concentration of 100 µM. Each toxin was added into the recirculated solution in a concentrationincreasing manner. Samples containing Karenia and Alexandrium cells (see details below) were perfused across the networks without recirculation. The networks were perfused with either the toxin or algal cell solutions at a flow rate of 1 mL/min until a new stable baseline of mean spike rate was established. Typically, each new baseline reached steady state within 10-20 min depending upon the toxin concentration. Toxin removal from a network was evaluated during the wash phase, where fresh recording solution was perfused over the networks without recirculation. Effects of the toxins on mean spike rate are presented as a percent of the control baseline level. The concentration giving 50% of the maximum inhibitory response or IC50 was calculated using logistical function with three parameters: A/Y ) ((1 + X)/X0)b where A is the mean spike rate in the absence of a toxin, Y is the mean spike rate in the presence of a toxin, X is the concentration of a toxin, X0 is the IC50, and “b” is the Hill coefficient obtained from the fit. Curve fitting was performed using Sigma Plot (version 8.0; SPSS Inc., Chicago, IL). Calculations of the detection limits were based on statistically significant changes, where P < 0.05 was considered significant as identified using either t-test or oneway ANOVA. Culturing of Karenia and Alexandrium, Cell Counts, Preparation of Isolates, and Application to Neuronal Networks. Dinoflagellate cultures were maintained as 25 mL batch cultures in Guillard’s f/2 medium with 10 µM selenium added. All cultures were incubated at 20 °C on a 16:8 light/dark cycle with a photon flux density of 75 µmol‚m-2s-1 (QSL 100; Biospherical Instruments, San Diego, CA). Cultures of Alexandrium fundyense (isolate GTCA-29, Portsmouth NH, Gulf of Maine) and Alexandrium affine (isolate CU-1) were generously provided by Dr. D. Anderson (Woods Hole Oceanographic Institute, MA). The Karenia brevis (K. brevis, isolate C-2,) and Karenia mikimotoi (K. mikimotoi, isolate NOAA-2, Sarasota, FL) cultures were generously provided by Dr. K. Steidinger (Florida Marine Research Institute, St. Petersburg, FL) and Dr. S. Morton (NOAA, Charleston, SC), respectively. Alexandrium cells were stained with Lugol’s iodine solution and counted under a microscope using a Sedgwick-Rafter counting chamber; Karenia cells were counted using a particle counter (Multisizer 3, Beckman Coulter, Miami, FL). Cultures were sonicated for 4 min on ice using a Branson probe sonicator (cat 101-063-200, VWR, USA) and inspected for complete cell lysis by light microscopy prior to neuronal network application. Sonicated algal VOL. 40, NO. 2, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Concentration-dependent effect of PbTx-3 on spinal cord neuronal networks. Recording trace of mean spike rate inhibition after introduction of various PbTx-3 concentrations. Inset panel (control experiment): concentration-dependency curve for ethanol (PbTx-3 vehicle) on spinal cord networks. The dashed line shows the range of ethanol concentrations used for PbTx-3 experiments; there is no statistically significant change in the network baselines after introduction of 1-10 µM ethanol. The error bars indicate standard deviations. samples were diluted to required concentrations with 2× solution (see above), water, and f/2 medium (where needed). Final volume of algal samples was 30 mL. PbTx Radioimmunoassay. PbTxs were extracted from the sonicated algal culture samples left from neuronal networks experiments in a separation funnel with 10 mL of methylene chloride followed by two 2.5 mL washes with methylene chloride. The methylene chloride fractions were combined and dried down with vacuum centrifugation in a SC210A Speedvac plus (Thermo Savant, Woburn, MA), then reconstituted in 1 mL of methanol.Values for PbTx content of K. brevis cultures expressed on a per cell basis were determined by radioimmunoassay as described by Woofter et al. (20). STX Receptor Binding Assay. Alexandrium cells were centrifuged followed by removal of the supernatant. The pellet was resuspended in 1 mL of 0.1N hydrochloric acid, sonicated for 1 min using a Branson probe sonicator, and boiled for 10 min. The resultant extract was centrifuged and filtered through a 0.2 µm PVDF filter. The pH of the extract was adjusted to pH 4.0 by the addition of 0.1 N sodium hydroxide prior to running the STX assay. The STX content of Alexandrium cultures expressed on a per cell basis was determined by receptor binding assay as described in Powell et al. (21).

Results The spinal cord neuronal networks on MEAs exhibited a mean spike rate of 45.5 ( 21.3 Hz (mean ( SD, n ) 11 networks, m ) 107 channels) and spike amplitude from 60 to 980 µV. Figure 1B shows 2 s of spontaneous spike activity on two individual channels in spinal cord neuronal network, showing the regularity in firing typical of in vitro spinal cord networks. Under control conditions, the change in mean spike rate from the original baseline was statistically insignificant for at least 8 h. Figure 2 shows the concentration-dependent effect of PbTx-3 on mean spike rate. Administration of PbTx-3 rapidly and reversibly inhibited the spike activity during constant perfusion. Dashed lines represent the introduction of various concentrations used in this experiment. The average recovery of mean spike rate after PbTx-3 exposures was 97 ( 2% (mean ( SD, n ) 3 networks) of the original level. The inset panel of Figure 2 shows the effect of ethanol, the solvent used for dissolution of PbTx-3, on the spinal cord network activity. The concentration-dependence curve of mean spike rate, 580

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FIGURE 3. Effect of various natural seawater dilutions on mean spike rate percent of control. The error bars indicate standard deviations (control-HEPES recording medium; n ) 3 networks; *p < 0.05; **p < 0.01). expressed as percent of control, was fitted to a 3 parameter logistic function yielding IC50, 250 ( 17 mM (mean ( SD, n ) 3 networks). It is important to note that during the PbTx-3 experiments, given the dilutions of the toxin used, the maximum concentration of ethanol reached only 0.01 mM such that the inhibitory effects on network activity could not be attributed to ethanol action. We next examined whether the neuronal networks could tolerate exposure to diluted natural seawater and algal culture medium (both ca. 30 ppt salinity). Application of either algal culture medium or seawater samples diluted at 1:25 v/v with HEPES recording medium (standard neuronal network recording medium) produced a 30-35% reduction in the mean spike rate: however, the networks were still sufficiently active with the newly established stable control baselines such that assays containing the algal culture medium/ seawater could be readily performed. Figure 3 summarizes the effects of various seawater dilutions on mean spike rate. Attempts to reduce the dilution of seawater to a level of 1:10 v/v led to total irreversible inhibition of mean spike rate of the networks in two out of four networks (data not shown). Concentration-dependent curves obtained for spiked seawater samples diluted more than 1:25 v/v (see Figure 3 for dilutions) were statistically indistinguishable from spiked HEPES recording medium samples for both PbTx-3 and STX (p < 0.05). Figure 4 illustrates the comparison of concentration-dependent effects of PbTx-3 (Figure 4A) and STX (Figure 4B) on mean spike rate in HEPES recording medium and 1:25 v/v diluted seawater. Under standard HEPES buffered conditions, PbTx-3 inhibited mean spike rate of the networks, with an average IC50 of 0.82 ( 0.12 nM (mean ( SD, n ) 3 networks). The detection limit for PbTx-3 in spinal cord networks corresponded to 0.33 ( 0.13 nM (mean ( SD, n ) 3 networks). The inhibition effect of PbTx-3 on neuronal networks in the presence of 1:25 v/v diluted seawater matrix was similar to that observed in HEPES recording medium. Average IC50 and detection limit in the presence of 1:25 v/v diluted seawater corresponded to 0.79 ( 0.17 nM and 0.48 ( 0.12 nM (mean ( SD, n ) 3 networks), respectively. Correlation coefficient between the curves with and without seawater corresponded to 0.937. STX inhibited mean spike rate with an IC50 of 0.13 ( 0.02 nM (mean ( SD, n ) 3 networks) in HEPES recording solution. The detection limit for STX in spinal cord neuronal networks corresponded to 0.031 ( 0.006 nM (mean ( SD, m ) 3 networks). On the other hand, various concentrations of STX, dissolved in 1:25 v/v diluted seawater, inhibited neuronal networks with an IC50 of 0.44 ( 0.06 nM and established the detection limit of 0.076 ( 0.016 nM (mean ( SD, n ) 3 networks). Correlation coefficient between the curves with and without seawater corresponded to 0.839. Figures 5 and 6 show examples of the effects of various algal samples on the spike activity from cultured neuronal

determined by radioimmunoassay and corresponded to 3.0 ( 0.7 pg/cell (mean ( SD, n ) 3). The small difference in the values reported for these two assays likely reflects, in part, different intrinsic activity of the brevetoxin congeners present in the algal cultures.

Discussion

FIGURE 4. Comparison of concentration-dependent inhibition of PbTx-3 (A) and STX (B) of mean spike rate in the presence of 25-fold diluted natural seawater. (b) Concentration-dependency curves in HEPES buffer and (2) in the presence of seawater (25-fold dilution). The points represent mean spike rate percent of control, and the error bars indicate standard deviations. Curves were fitted with 3 parameter logistical function. Correlation coefficients between two curves in each panel corresponded to 0.937 (A) and 0.839 (B). networks. Due to high concentration of the toxins in algal samples they were diluted at least 100 times. Figure 5A shows rapid and reproducible inhibition of the mean spike rate with two consecutive applications of STX-producing Alexandrium fundyense (GTCA-29) cell samples at concentrations ca. 93 cell/mL. Samples of this Alexandrium isolate (ca. 9300 cell/mL) were sonicated and diluted 1:100 with HEPES recording medium. The inhibitory effect of GTCA-29 was completely eliminated by washing with diluted 1:100 v/v f/2 cell culture growth medium. Figure 5B demonstrates that introduction of the non-STX isolate Alexandrium affine (CU1) at final concentration ca. 2.5 cell/mL failed to induce a response from the neuronal networks, in marked contrast to the effect of GTCA-29 when it was used at the same cell concentration (ca. 2.5 cell/mL). A similar trend is also shown in Figure 5C, which summarizes the mean effect of CU-1 and GTCA-29 on mean spike rate at concentrations ca. 93 cell/ mL. According to concentration-dependence curves for STX (Figure 4B) and effect on mean spike rate for GTCA-29 algal samples (Figure 5A, B, C), the amount of STX (based on diacetate equivalent) per cell corresponded to 9.7 ( 0.8 pg/ cell (mean ( SD, n ) 4 networks). The amount of STX per cell in the sample was also measured by receptor binding assay (see method section) and corresponded to 45 ( 26 pg/cell (based on dihydrochloride equivalent). Figure 6A shows the recording trace of mean spike rate inhibition during the exposure of the neuronal network to K. mikimotoi (non-PbTx producer, NOAA-2 isolate) and K. brevis (PbTx producer, C-2 isolate) algal samples at final concentrations of ca. 400 cell/mL. Figure 6B shows the summary of the effects of both types of Karenia cells on mean spike rate inhibition, which corresponded to 98 ( 2% (p < 0.001) and 11 ( 5% (p < 0.05) (mean ( SD, n ) 3 networks) for K. brevis and K. mikimotoi, respectively. According to concentration-dependence curves for PbTx-3 (Figure 4A) and results obtained from the effect of K. brevis algal samples on mean spike rate (Figure 6), the amount of PbTx (based on PbTx-3 equivalent) per cell corresponded to 6.8 ( 1.7 pg/cell (mean ( SD, n ) 3 networks). The amount of PbTx-3 equivalents per cell in the sample was also

In this work, we examined the utility of the NNB for the detection of important HAB neurotoxins in algal samples, where a minimum amount of sample preparation was evaluated. We demonstrated that the neuronal networks are sufficiently robust to tolerate administration of diluted natural seawater samples with virtually no change in the nanomolar sensitivity to the marine toxins. Both PbTx-3 and STX are powerful neurotoxins that exhibit distinct actions on VGSCs. PbTx-3 enhances activation of the VGSC and prevents its closing, leading to energy depletion and loss of neuronal activity (22-24). In contrast, STX is a VGSC inhibitor that prevents propagation of action potentials in neurons (25). In this study, both STX and PbTx-3 inhibited mean spike rate of spinal cord neuronal networks within minutes of exposure, despite the distinct actions of these algal neurotoxins on nervous tissue. These observations on spike dynamics of spinal cord networks agree with previously demonstrated effects of STX and PbTx-2 on networks derived from frontal cortex cells (15). STX and PbTx-2 produced distinctive effects on electrophysiological parameters such as burst properties, but not spike dynamics. These results correlate well with the effects at similar concentrations reported for other types of function-based assays (6, 8, 10). Spinal cord networks showed high sensitivity to both STX and PbTx-3, with inhibition occurring at sub-nanomolar concentrations. From these results, a detection limit of 31 pM can be derived, which is equivalent to 0.012 ppb of STX (based on STX diacetate). This value is 30 000 times below the mouse bioassay detection limit. Although the spinal cord networks are an order of magnitude less sensitive to PbTx-3 than to STX, the detection limit is still approximately 300 times below the regulatory limit (0.8 ppm). Like any cellular assay, neuronal network function can be influenced by perturbations in ionic constituents of the extracellular medium. Environmental samples containing natural seawater or algal culture medium could not be introduced to neuronal networks before significant reduction of salt concentrations. Due to great sensitivity of spinal cord neuronal networks to both STX and PbTx-3, simple dilution could be applied to minimize matrix effects without the need for complex sample preparation. Although in this study we used 1:100 v/v dilutions of natural seawater samples, the neuronal networks exhibited stable baseline activity with 1:25 v/v dilution. A major advantage of sample dilution is that this method could be readily implemented in an automated format with low-power pumps, mixers, and valves. Nevertheless, dilution could result in a loss of detection sensitivity. Alternative methods for sample processing include the use of dynamic dialyzers to remove salt contaminants and avoid dilution. One can make some reasonable approximations of the expected levels of PbTx in culture or during a bloom event. Assuming a K. brevis bloom concentration ranging from 105 to 106 cells/L (10-100 pg/cell of PbTx), the resulting concentration range is approximately 1-100 nM. Given 1:25 v/v dilution of original sample concentration range becomes 0.04-4 nM. Since it appears possible to detect 0.3 nM PbTx-3 with the NNB, detection during a bloom event can be accomplished by direct examination of sonicated seawater samples. Due to even higher sensitivity of neuronal networks to STX, various dilutions could be applied to the toxin containing samples depending on HAB intensity without significant loss of detection ability. VOL. 40, NO. 2, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 5. (A) Effects of sonicated Alexandrium fundyense (GTCA-29, STX producer) cell samples at 100-fold dilution on spinal cord neuronal networks (final concentration ca. 93 cell/mL). Effect of GTCA-29 was eliminated by washing with f/2 growth medium (100-fold dilution). (B) The recording trace of mean spike rate during introduction of Alexandrium affine (CU-1, non-STX producer) and Alexandrium fundyense (GTCA-29) at 100-fold dilution and final sample concentrations ca. 2.5 cell/mL. (C) Effects of Alexandrium CU-1 and GTCA-29 (ca. 93 cell/mL) on mean spike rate percent of control. The error bars indicate standard deviations with **p < 0.001 and n ) 3 networks. Neuronal networks were not affected by the presence of non-STX producing algae. However, K. mikimotoi that is nonbrevetoxin isolate surprisingly caused a small but significant effect on neuronal network baseline. According to recent reports K. mikimotoi was associated with fish kills due to the production of yet-uncharacterized compounds such as polyunsaturated fatty acids (29) and gymnocins (30) where the former has a potential to modulate neuronal activity (31). Nevertheless, these findings indicate that equivalent numbers of cells from K. brevis produce a much more potent inhibition of network activity than cells from K. mikimotoi, an observation consistent with the known relative toxicities of these algal species.

FIGURE 6. (A) Comparison of the effects of sonicated K. mikimotoi (non-PbTx producer) and K. brevis (C-2, PbTx-3 producer) cell samples on spinal cord neuronal networks at final sample concentration ca. 400 cell/mL (original samples were diluted 100 fold). Effect of K. brevis was eliminated by washing with f/2 growth medium (100-fold dilution). (B) Effect of Karenia cells on mean spike rate percent of control. The error bars indicate standard deviations with *p < 0.05 and **p < 0.001, n ) 4. Other in vitro assays for the detection of marine toxins that use either cells or tissue slices have been described in the literature (6-8, 10, 21, 26, 27). Although they all show some sensitivity to marine toxins, the major advantage of the neuronal networks is the relative robustness, exemplified by the fact that shipping can be accomplished by commercial carrier (19, 28). The use of a common assay format would allow for consistency among laboratories and raises the possibility of use the cultured neuronal networks in the field. 582

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The strength of the use of physiologically based detection approaches, such as cultured neuronal networks, is in detection of samples that have biologic activity. In addition, physiologically based detection approaches are integrative and biologically active mixtures can be detected. Although neuronal networks could be used to classify potential neurotoxins due to their signature effects on electrophysiological parameters (15), it is important to recognize that this generic detection approach will neither fully identify nor quantify the individual toxins. Therefore, generic detection will not replace structure-based assayssrather it can be complementary. We anticipate that samples that induce a positive response from generic detection approaches can be further processed using structure-based assays that not only identify the toxin, presuming it is known, but also quantify the concentration. This study demonstrated that, based on neuronal networks cultured over MEAs, portable NNB is capable of the sensitive and rapid screening of STX and PbTx-3 dissolved in seawater or associated with their algal producers. Results obtained here for the amount of toxin show some agreement with concentrations determined by alternative methods such as receptor binding assay and radioimmunoassay. NNB technology is not limited to these two marine toxins and algal taxa. In fact, our other studies showed that neuronal network-based assays could be used for monitoring domoic acid and azaspiracid at concentrations that may be found in

environmental samples including shellfish extracts (unpublished). Moreover, as a function-based tool, NNB could find application for detection of yet-uncharacterized neurotoxins.

Acknowledgments This work was supported by the Defense Advanced Research Projects Agency and the National Oceanic Atmospheric Administration (NOAA). N.V.K. holds a National Research Council Research Associateship at the Naval Research Laboratory. The authors gratefully acknowledge the technical contribution from Dr. Thomas O’Shaughnessy and the assistance of Ricky Woofter with the radioimmunoassay analysis.

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Supporting Information Available The opinions and assertions contained herein are those of the authors and are not to be construed as official or reflecting the view of the Department of the Navy. This publication does not constitute an endorsement of any commercial product or intend to be an opinion beyond scientific or other results obtained by NOAA. No reference shall be made to NOAA, or this publication furnished by NOAA, to any advertising or sales promotion which would indicate or imply that NOAA recommends or endorses any proprietary product mentioned herein, or which has as its purpose an interest to cause the advertised product to be used or purchased because of this publication.

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Received for review July 1, 2005. Revised manuscript received November 10, 2005. Accepted November 15, 2005. ES051272A

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