Vol. 290, Issue 1, 439-444, July 1999

Brevetoxins Cause Acute Excitotoxicity in Primary Cultures of Rat Cerebellar Granule Neurons¹

Frederick W. Berman and Thomas F. Murray

Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia

Brevetoxins (designated PbTx-1 to -10) are potent lipid-soluble polyether compounds that are known to bind to and modulate voltage-gated sodium channel activity. To investigate whether brevetoxins produce direct central nervous system neurotoxic effects, cultured rat cerebellar granule neurons were exposed to brevetoxins in Locke's buffer for 2 h at 22°C. Neuronal injury was quantified by assaying lactate dehydrogenase activity in the exposure buffer and in conditioned growth media collected at 22 h after brevetoxin exposure. Brevetoxins produced acute neuronal injury and death in neurons with a rank order potency of PbTx-1 (EC₅₀ = 9.31 ± 0.45 nM) > PbTx-3 (EC₅₀ = 53.9 ± 2.8 nM) > PbTx-2 (EC₅₀ = 80.5 ± 5.9 nM) > PbTx-6 (EC₅₀ = 1417 ± 32 nM), which is similar to their previously determined rank order potency for brevetoxin-induced icthyotoxicity and binding to [³H]PbTx-3-labeled sodium channels on synaptosomes. The neurotoxic response could be prevented by coapplication of the sodium channel antagonist tetrodotoxin or by the competitive or noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists D-AP5 and MK-801, ketamine, dextromethorphan, and dextrorphan, respectively. NMDA receptor antagonists afforded neuroprotection with rank order potencies comparable to those measured previously for protection against glutamate-induced excitotoxic responses. Further analysis revealed that brevetoxins induced a concentration-dependent release of L-glutamate and L-aspartate into the exposure buffer. These data indicate that brevetoxin-induced injury in cultured rat cerebellar granule neurons is mediated by NMDA receptors that are activated indirectly as a consequence of PbTx-induced sodium channel activation and attendant excitatory amino acid release.