Neurotoxin-like compounds from the ichthyotoxic red tide alga Heterosigma akashiwo induce a TTX-like synaptic silencing in mammalian neurons

Heterosigma akashiwo (Raphidophyceae) is a golden-brown unicellular marine microalgae that has been implicated in massive numbers of fish deaths in natural and cultured populations around the world. The ichthytoxicity of this species has been associated to the presence of a neurotoxin-like compound. The aim of this work was to study the neurotoxic properties of an extract of H. akashiwo. Under our experimental conditions, the H. akashiwo microalgae induced a paralytic effect on the mobility of Artemia salina in a concentration-dependent manner, abolishing the natatorial capacity of A. salina. Similarly, this capacity was reduced with the presence of toxin extract from H. akashiwo (HaTx, 41 ± 8%) in the media. Additionally, using a neuroblastoma cell based assay, HaTx showed a concentration-dependent decrease in cytotoxicity in the presence of ouabain/veratridine (30 ± 3%). Using patch clamp and Ca2+ microfluorimetry in hippocampal neuron cultures, we measured the acute effects of HaTx on neuronal network activity. HaTx (0.5% v/v) decreased the frequency (control 16 ± 3 Hz vs HaTx 7 ± 1.7 Hz) and amplitude (control 315.8 ± 9.7 pA vs 118.6 ± 2.2 pA) of synaptic electrophysiological activity and also reduced intracellular Ca2+ transient frequency (control 4.5 ± 0.2 Hz vs HaTx 2.1 ± 0.3 Hz). Both types of events were associated with synaptic silencing and synaptic dyshomeostasis. HaTx toxicity was reversible, recovering about 70% of neuronal activity when the toxin was withdrawn. Furthermore, immunostaining of SV-2, a key secretory protein, revealed a significant decrease suggesting synaptic remodeling. In conclusion, HaTx demonstrated cellular and neuronal toxic properties that were mediated by a fast and reversible voltage-dependent sodium channel blockade, and could be a new interesting neuroactive compound having pharmacological benefits.