Azaspiracid-1 inhibits bioelectrical activity of spinal cord neuronal networks Nadezhda V. Kulagina a, * , Michael J. Twiner b , Philipp Hess c , Terry McMahon c , Masayuki Satake d , Takeshi Yasumoto e , John S. Ramsdell b , Gregory J. Doucette b , Wu Ma a , Thomas J. O’Shaughnessy a a Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue, SW, Code 6900, Washington, DC 20375, USA b Marine Biotoxins Program, Center for Coastal Environmental Health and Biomolecular Research, NOAA/National Ocean Service, 219 Fort Johnson Road, Charleston, SC 29412, USA c Biotoxin Chemistry, Marine Environment and Food Safety Services, Marine Institute, Galway Technology Park, Parkmore West, Galway, Ireland d Graduate School of Agricultural Science, Tohoku University, Sendai, Japan e Japan Food Research Laboratory, Tama Laboratory, Nagayama, Tama, Tokyo, Japan Received 17 August 2005; revised 13 December 2005; accepted 7 February 2006 Available online 19 April 2006 Abstract Azaspiracid-1 (AZA-1) is a recently identified phycotoxin that accumulates in molluscs and can cause severe human intoxications. For this study, we utilized murine spinal cord and frontal cortex neuronal networks grown over 64 channel microelectrode arrays (MEAs) to gain insights into the mechanism of action of AZA-1 on neuronal cells. Extracellular recordings of spontaneous action potentials were performed by monitoring mean spike rate as an assay of the efficacy of AZA-1 to alter the bioelectrical activity of neurons in the networks. Via slow onset, AZA-1 decreased the mean spike rate of the spinal cord neurons with an IC 50 of ca. 2.1 nM, followed by partial recovery of original activity when toxin was removed. Pre- treatment with the GABA A receptor antagonist bicuculline led to an increased response of the neuronal networks to AZA-1 exposure and resulted in an irreversible inhibition of spike rate. AZA-1 did not cause any changes in frontal cortex networks upon drug exposure. In addition, whole-cell patch clamp recordings from spinal cord neurons showed that AZA-1 had no significant effect on the voltage-gated sodium (Na C ) or calcium (Ca 2C ) currents, suggesting that the toxin affected synaptic transmission in the neuronal networks through a mechanism independent of these voltage-gated channels. Crown Copyright q 2006 Published by Elsevier Ltd. All rights reserved. Keywords: Biosensor; Azaspiracid-1; Extracellular recording; Phycotoxin; Microelectrode array; Primary neuronal cultures; Voltage-gated channels; Action potential 1. Introduction In 1995, a novel marine phycotoxin, azaspiracid (AZA), was identified following cases of human intoxication in the Netherlands resulting from the consumption of mussels cultivated in Killary Harbour, Ireland (McMahon and Silke, 1996). The illness was subsequently termed azaspiracid shellfish poisoning (AZP). Since then cases of AZA intoxication and/or contamination of shellfish have been documented in several other European countries including UK, Norway, France, Spain and Italy (Satake et al., 1998; James et al., 2002; Magdalena et al., 2003). Toxicon 47 (2006) 766–773 www.elsevier.com/locate/toxicon 0041-0101/$ - see front matter Crown Copyright q 2006 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2006.02.011 * Corresponding author. Fax: C1 202 767 9594. E-mail address: kulagina@cbmse.nrl.navy.mil (N.V. Kulagina).