Elsevier Editorial System(tm) for Harmful Algae Manuscript Draft Manuscript Number: HARALG-D-15-00044 Title: NEUROTOXIN-LIKE COMPOUNDS FROM THE ICHTHYOTOXIC RED TIDE ALGA HETEROSIGMA AKASHIWO INDUCE A TTX-LIKE SYNAPTIC SILENCING IN MAMMALIAN NEURONS Article Type: Original Research Article Keywords: Heterosigma akashiwo; Raphidophyte; Na-channel blockers; TTX-like compounds; harmful algal bloom; synaptic silencing. Corresponding Author: Dr. Jorge Fuentealba, PhD Corresponding Author's Institution: University of Concepcion First Author: Allisson Astuya, PhD Order of Authors: Allisson Astuya, PhD; Alejandra E Ramirez, Biochemistry; Ambbar Aballay, Engineer on Biotechnology; Juan Araya, Biochemistry; Janette Silva, MSc; Viviana Ulloa, PhD; Jorge Fuentealba, PhD Abstract: 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 ictiotoxicity 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 Heterosigma akashiwo microalgae induced a paralytic effect on the mobility of Artemia salina in a concentration-dependent manner, abolishing the natatorial capacity of A. salina. in the same way, this capacity was reduced with the presence on the media of toxin extract from Heterosigma akashiwo (HaTx, 41 ± 8%). Additionally, using a neuroblasto 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 HaTX toxicity was reversible, recovering about 70% of neuronal activity when the toxin was withdrawn.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. Suggested Reviewers: Luis Gandia PhD Professor, Pharmacology, Universidad Autonoma de Madrid luis.gandia@uam.es Dr. Gandia is an expert on electrophysiology and ionic channels Hermes Yeh PhD Professor, Physiology and Neurobiology, Geisel School of Medicine At Dartmouth

hermes.yeh@dartmouth.edu in an expert in the fiel of neurobiology Terrance Egan PhD Professor, Pharmacological and Physiological Science, Saint Louis University egantm@slu.edu He is an expert on receptors and ionic channles

Concepcion, March 04, 2015

Dr.

Sandra E. Shumway

Editor

Harmful Algae Journal

Dear Dr. Shumway

We are submitting a manuscript entitled “Neurotoxin-Like Compounds From

The Ichthyotoxic Red Tide Alga Heterosigma Akashiwo Induce A Ttx-Like

Synaptic Silencing In Mammalian Neurons” to be considered for publication in

Harmful Algae Journal. The authors are Allisson Astuya, Alejandra E Ramírez, Ambbar

Aballay, Juan Araya, Janette Silva, Viviana Ulloa and myself.

As we commented to you in our enquiry sent in the last Nov 13, 2014, this work

provides new evidence related with the characterization of an extract obtained from

heterosigma akashiwo that have TTX like properties. We have characterized this

extract by in vivo test, neuronal electrophysiology, calcium signals and

innmunocytochemistry.

The submitted Manuscript has not been nor will be submitted to any other

journal if accepted for publication in the Harmful algae

Yours sincerely,

Jorge Fuentealba, PhD

Associated Professor

Cover Letter

NEUROTOXIN-LIKE COMPOUNDS FROM THE ICHTHYOTOXIC RED TIDE ALGA

HETEROSIGMA AKASHIWO INDUCE A TTX-LIKE SYNAPTIC SILENCING IN

MAMMALIAN NEURONS

Allisson Astuyaa, Alejandra E Ramírezb, Ambbar Aballaya, Juan Arayab, Janette Silvac,

Viviana Ulloaa, Jorge Fuentealba b, d*

Highlights: Our recent work are related with the characterization of an extract obtained

from Heterosigma akashiwo that have TTX like properties. We have characterized this

extract by in vivo test, neuronal electrophysiology, calcium signals and

innmunocytochemistry.

Keywords: Heterosigma akashiwo; Raphidophyte; Na-channel blockers, TTX-like

compounds, harmful algal bloom, synaptic silencing.

Corresponding Author: Jorge Fuentealba Arcos, PhD., Screening of Neuroactive

Compounds Unit, Department of Physiology, Faculty of Biological Sciences, University of

Concepcion, Barrio Universitario s/n, Concepcion, Chile, POBOX 160-C; Phone: 56-41-

2661082; Fax: 56-41-2204557; email: jorgefuentealba@udec.cl

*Highlights (for review)

NEUROTOXIN-LIKE COMPOUNDS FROM THE ICHTHYOTOXIC RED TIDE ALGA

HETEROSIGMA AKASHIWO INDUCE A TTX-LIKE SYNAPTIC SILENCING IN

MAMMALIAN NEURONS

Allisson Astuyaa, Alejandra E Ramírezb, Ambbar Aballaya, Juan Arayab, Janette Silvac,

Viviana Ulloaa, Jorge Fuentealba b, d*

a Laboratory of Cell Culture and Marine Genomics, Marine Biotechnology Unit, Faculty of Natural and

Oceanographic Sciences and COPAS Sur-Austral Program. University of Concepcion, Concepción, Chile.

b Screening of Neuroactive Compounds Unit, Department of Physiology, Faculty of Biological Sciences.

University of Concepcion, Concepción, Chile.

c Laboratory of Bioassay, Department of Zoology, Faculty of Natural and Oceanographic Sciences, University

of Concepción, Chile.

d Unit for Pharmacological Development, Center of Biomedicine, University of Concepcion, Chile.

Keywords: Heterosigma akashiwo; Raphidophyte; Na-channel blockers, TTX-like

compounds, harmful algal bloom, synaptic silencing.

Corresponding Author: Jorge Fuentealba Arcos, PhD., Screening of Neuroactive

Compounds Unit, Department of Physiology, Faculty of Biological Sciences, University of

Concepcion, Barrio Universitario s/n, Concepcion, Chile, POBOX 160-C; Phone: 56-41-

2661082; Fax: 56-41-2204557; email: jorgefuentealba@udec.cl

*ManuscriptClick here to view linked References

ABSTRACT

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 ictiotoxicity 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

Heterosigma akashiwo microalgae induced a paralytic effect on the mobility of Artemia

salina in a concentration-dependent manner, abolishing the natatorial capacity of A. salina.

in the same way, this capacity was reduced with the presence on the media of toxin

extract from Heterosigma akashiwo (HaTx, 41 ± 8%). Additionally, using a neuroblasto 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.

INTRODUCTION

The Raphidophyceae class includes unicellular algae involved in massive fish mortalities

in wild and cultured populations producing substantial losses in the aquaculture industry

on a worldwide scale (Kim et al., 2007; Landsberg, 2002). Fibrocapsa japonica,

Chattonella spp. and Heterosigma akashiwo are some of the main ichthyotoxic species

pertaining to this class (Fu et al., 2004; Landsberg, 2002) and have been involved in

harmful algal blooms (HABs) in every ocean around the world (Bowers et al., 2006;

Connell, 2000; Landsberg, 2002).

Although it has been postulated that the highest toxicity of rhaphidophytes occurs in the

exponential phase of growth, the toxicological mechanism(s) responsible for the

ichthyotoxic properties of H. akashiwo are currently under debate and three main

mechanisms have been proposed: i) an elevated production in reactive oxygen species

(ROS) that are normally generated during the growth of these microalgae (Kim et al.,

2007; Oda et al., 1997) and have been linked to fish gill tissue injury such as epithelial

lifting, cell necrosis and alteration of chloride cells, producing massive mucus secretion

from the gills and physiological responses such as hypoxia and subsequent asphyxia (Kim

et al., 2000; Marshall et al., 2005; Twiner et al., 2001); ii) a high content of free fatty acids

(FFAs) (Fu et al., 2004; Ling and Trick, 2010; Marshall et al., 2003) which have been

associated with hemolytic activity in fish blood cells (de Boer et al., 2009; Pezzolesi et al.,

2010); and iii) an association between rhaphidophytes toxicity and the production of an

organic toxin, such as neurotoxins or cardiotoxins, which can lead to respiratory and/or

cardiac paralysis and abnormal behavior (Endo et al., 1992; Khan et al., 1997; Landsberg,

2002).

It has been proposed that rhaphidophytes toxins could be brevetoxin or brevetoxin-like

compounds and this has been found in both laboratory cultures of H. akashiwo and in

water containing blooms of H. akashiwo (Khan et al., 1997). Nevertheless, several authors

have demonstrated low concentrations or even the lack of these neurotoxin-like

compounds (de Boer et al., 2012; Khan et al., 1996; Lewitus and Holland, 2003). On the

other hand, a synergistic role of more than one mechanism of toxicity has been postulated

(Marshall et al., 2003) complicating the search for the real compounds associated with the

toxic mechanism of H. akashiwo. In order to elucidate the nature of the neuroactive

compounds associated with the toxicity of H. akashiwo, we decided to do a functional

characterization of the toxins present in ichthyotoxic microalgae crude extracts since many

marine toxins have been described to bind ion channels (Blumenthal, 1995), specifically

voltage-gated sodium channels (VGSC) (Catterall, 1980; Catterall and Gainer, 1985;

LePage et al., 2003; Wang and Wang, 2003). Therefore, the objective of this work was to

characterize the effects of a crude extract from H. akashiwo in vivo and in vitro to further

our understanding of the mechanism of action of the toxins present in this species.

MATERIALS AND METHODS

Algal strains and culture maintenance

Heterosigma akashiwo (CCMP302, New Zealand) was used for this study. The microalga

was obtained from the Provasoli-Guillard National Center for Culture of Marine

Phytoplankton, Maine, USA (CCMP). The green unicellular marine alga Tetraselmis

tetrathele was obtained from the Microalgae Culture Collection CCM-UdeC at the

Universidad de Concepción, Chile and was used as a non-toxic control, considering the

similar cellular size compared with H. akashiwo.

Both strains were maintained under similar experimental conditions in 1L Erlenmeyer

flasks containing 500 mL of L1 medium (Guillard and Hargraves, 1993). 10,000 cells/mL

were inoculated from acclimated, exponential-phase stock cultures. Cultures were

incubated in a culture chamber at 15 ± 2°C, 50 μmol photon m-2s-1 and a 16:8 (L:D)

photoperiod, without aeration, but were shaken manually once per day. Cell density was

assessed by cell counting in 1 mL Utermöhl chambers.

Preparation of toxin extract from H. akashiwo (HaTX)

For toxin extraction, H. akashiwo microalga at 181,000 cells/mL in the exponential growth

phase was harvested through filtration using Millipore 0.45 µm filters. Filters were

sonicated during 10 min at 47 KHz ± 6% (Branson 1210, Connecticut, USA) in absolute

methanol with an extraction volume of 11 mL and the volume was reduced to 1/6 parts

using a rotary evaporator (IKA, USA) at 40°C. One mL of crude extract was loaded on

reversed-phase SPE cartridges ODS-C18 (500 mg, 6 mL; Accuboound II, UK), and toxins

were eluted with 3 mL methanol. The final methanolic stock solution containing

approximately 0.55 mg/ml was stored in darkness at -20°C.

Artemia salina bioassay

Artemia salina cysts were hatched in filtered marine water (S‰=35 g/L) two days before

initiating the test at 25 ºC. Five larvae instar II-III were transferred into each well in 10 mL

of different H. akashiwo culture densities for a 24 h period. Four replicates for each

treatment were established. Four bioassays were made with H. akashiwo in the

exponential growth phase. The culture densities evaluated in each bioassay were 4,690;

9,375; 18,750; 37,500 and 75,000 cells/mL. Controls with Tetraselmis tetrathele at 75,000

cells/mL was used considering the highest concentration of H. akashiwo. The effect of H.

akashiwo on the Artemia larvae was observed under a dissecting scope (Olympus TL2,

Japan). The results were represented as percentage of larvae able to move through the

water (percentage of larvae mobility) after 24 h exposure.

Neuroblastoma Neuro2a cytotoxicity assay

Mouse neuroblastoma Neuro-2a cells (ATCC, CCL-131) were maintained in 10% fetal

bovine serum (FBS) RPMI 1640 medium (Gibco-Life Technologies, USA) at 37 ºC in a 5%

CO2 humid atmosphere (Memmert GmbH & Co. KG, Germany). For the experiments, cells

were seeded in a 96-well microplate in 5% FBS RPMI 1640 medium at an approximate

density of 35,000 cells per well and incubated 24 h before exposure in the same conditions

described for cell maintenance. In order to detect the presence of neurotoxins acting on

VGSCs, the Neuro-2a cytotoxicity bioassay was performed. Neuro-2a cells were treated

with the Na+/K+-ATPase inhibitor ouabain (0.3 mM), the sodium channel-activator

veratridine (0.03 mM) and HaTX (0.14-4.35 %v/v). The reduction of cytotoxicity in the

presence of ouabain/veratridin or “cell rescue" is indicative of a sodium channel blocking

toxin (paralyzing toxins). Additionally, the enhancement of cytotoxicity in the presence of

ouabain/veratridine is indicative of a potent sodium channel activator (brevetoxin-like

toxins) (Manger et al., 1993; Hayashi et al., 2006; Cañete and Diogène, 2008; Caillaud et

al., 2010). To discard cytotoxicity of the H. akashiwo extract, independent of the VGSC-

effect, Neuro-2a cells were exposed to the extract in the absence of ouabain/veratridine.

Cell viability was measured after 24 h incubation using the colorimetric MTT assay

(Manger et al., 1993). Absorbances were read at 570 nm using an automated multi-well

scanning spectrophotometer (Synergy H1, Biotek, Vermont, USA). The extract effect was

represented as percentage of cell rescue.

Hippocampal cultures

18-19 days pregnant Sprague-Dawley rats were treated in accordance with regulations

established by NIH and the Ethics Committee at the University of Concepción. Rat

embryonic hippocampi were separated and plated at 250,000 cells/ml on coverslips coated

with poly-L-lysine (Sigma Aldrich, St. Louis, MO, USA). Cultures were maintained at 37 ºC

with 5% CO2. Culture medium was replaced every 3 days and consisted of 90% minimal

essential medium (MEM; Gibco, Grand Island, NY, USA), 5% heat-inactivated horse

serum, 5% fetal bovine serum (FBS) and N3 supplement (mg/ml: BSA 1, Putrescine 3.2,

Insulin 1, Apotransferrin 5, Corticosterone 0.5; g/ml: Sodium selenite 5, TH3 0.5,

Progesterone 0.6). Experiments were performed at 10–12 DIV (days in vitro) in control and

treated neurons.

Electrophysiology

Spontaneous synaptic currents were recorded in hippocampal neurons during 2 minutes

using the patch clamp technique (Axon amplifier 200B, Molecular Devices) in voltage

clamp mode (-60 mV holding potential) and whole cell configuration. The pipette solution

contained (in mM): 140 KCl, 10 BAPTA, 10 HEPES, 4 MgCl2, 0.3 GTP and 2 ATP-Na2, at

pH 7.4 and 300 mOsm. After basal synaptic recording, neurons were perfused for 30 s

with TTX (0.25 nM) and/or HaTX (0.5 %v/v) in normal external solution, using a fast

perfusion system. Before and after the perfusion, cells were washed with normal external

solution. Amplitude and frequency of miniature postsynaptic currents (mPSC) were

analyzed with the Minianalysis program (Synaptosonft Inc.). Miniature excitatory or

inhibitory post-synaptic currents (mEPSC or mIPSC, respectively) were pharmacologically isolated

with TTX (50 nM) and bicuculline (5 µM) for mEPSC, or TTX (50 nM), D-AP5 (50 µM), CNQX (5 µM)

for mIPSC. The electrophysiological activity was recorded for 2 min with the whole-cell patch

clamp technique as previously published (Fuentealba et al., 2012), using an Axopatch 200B

amplifier (Axon Instruments, Sunnyvale, CA, USA) in voltage clamp mode (holding potential -60

mV).

Spontaneous Ca2+ transients

Spontaneous Ca2+ transients were measured using the calcium-sensitive dye Fluo4-AM®

(3 µM, Invitrogen, USA). Hippocampal neurons were placed on coverslips coated with

poly-L-lysine (Sigma-Aldrich) and incubated with the dye for 30 min at 37 ºC. The neurons

were then washed twice and mounted on a perfusion chamber on a microscope (Nikon,

TE 2000, Japan). The Fluo4-AM® was excited with a band pass filter at 480 nm wave-

length and the emission was detected at 535 nm. Changes in cytosolic Ca2+ were

registered at different times of incubation with TTX (0.25 nM) or HaTX (0.5 %v/v) using an

EM-CCD camera (iXon ANDOR, USA) and a Lambda 10-B interface (Sutter Instruments,

USA). The washout effects were registered after toxin solution replacement with external

solution. Each image was recorded every 0.5–1 s with a time exposure of 200 ms. Image

analyses were made selecting representative regions of interest (ROIs) in cytosolic

regions and using the Imaging Workbench 6.0 software (Indec System, USA).

Immunocytochemistry

Control and treated (similar conditions to Ca2+ experiments) hippocampal neurons were

fixed for 10 min with methanol, and permeabilized with 0.1% Triton X-100 in phosphate-

buffered saline (PBS). Nonspecific immunoreactivity was blocked with 10% horse serum

for 30 min at room temperature. Monoclonal anti-SV2 antibody (1:200, Developmental

Studies Hybridoma Bank, Iowa City, USA) and polyclonal anti MAP2 (1:200, Santa Cruz

Biotechnology, CA, USA) antibody were incubated overnight followed by incubation with

an anti-mouse secondary antibody conjugated with AlexaFluor 488 (1:200; Jackson

ImmunoResearch Laboratories, USA) and anti-rabbit secondary antibody conjugated with

Cy3 (1:200; Jackson ImmunoResearch Laboratories, USA), respectively, for 1 h at room

temperature. The samples were mounted in fluorescent mounting medium (DAKO, USA)

for microscopic analysis. An LSM710 confocal microscope (Axio Imager.Z1, Carl Zeiss,

Germany) with ZEN 2008 software v5.0 (Zeiss) was used to acquire multi-channel

fluorescence images (63X, NA 1.4, oil immersion) with lasers of Argon (488 nm) and He-

Ne (543 nm) for Alexa Fluor 488 and Cy3, respectively.

Data Analysis

Data are presented as mean ± SE. Statistically significant differences were considered

with P<0.05. Differences in the mobility assay were determined using Dunnett paired

parametric test on ToxStat Software (University of Wyoming, USA). Results of Neuro-2a

cytotoxicity assay, electrophysiological and imaging experiments were analyzed with

GraphPad Prism version 4 (GraphPad Software, California, USA).

RESULTS

Heterosigma akashiwo induces an inhibition of Artemia salina mobility

Artemia salina larvae are characterized by their swimming ability in sea water. In

this study, the presence of H. akashiwo for 24 h induced a significant reduction in

the mobility capacity of A. salina that was dependent on the microalgae density

(4,690-18,750 cells/ml), with mobility being completely abolished (1.7 ± 3.5 %,

n=120) when higher densities of H. akashiwo (37,500-75.000 cells/ml) were used

(Fig. 1A). Although the larvae lost their swimming ability, or mobility, they

maintained movement of their swimming appendages. To discard the possibility

that H. akashiwo density induced a change on the viscosity of sea water, a non-

toxic microalga (T. tetrathele 75,000 cells/ml) was used. No differences on mobility

were observed in the presence or absence of T. tetrathele (data not shown). These

results suggest that H. akashiwo releases some bioactive or toxic compound that

interferes with the motor function of A. salina. Following this assumption, an extract

obtained from H. akashiwo (HaTX) was tested in the A. saline bioassay. The HaTx

extract (5% v/v, 24 h) induced a decrease on the mobility of A. saline about 42 ±

14% (n=80, Fig. 1B), reinforcing the idea that this effect is mediated by the

presence of some bioactive compound in the extract.

HaTx reverts the cytotoxicity of ouabain/veratridine on Neuro2a cells

As mentioned above, some species of Raphidophyceae are known to possess

neurotoxic compounds; therefore, we used a cellular approach to evaluate the

potential modulation of sodium channels. The assay was performed using Neuro2A

cells to assess the presence of toxins having sodium channel activation capacity

(brevetoxin-like compounds) or sodium channel blocking activity (such as saxitoxin

or tetrodotoxin). No significant brevetoxin-like activity was detected in the HaTX

extract (data not shown). Moreover, as shown in Figure 2, Neuro-2a cells exposed

for 24 h to a range of HaTX concentrations (0.14 - 4.35 %v/v) showed a

concentration-dependent reduction of cytotoxicity in the presence of

ouabain/veratridine (cell rescue), eliciting about 30 ± 3% of viability recovery at

higher concentrations (4.35 %v/v). These results suggest that HaTX antagonizes

the effect induced by ouabain/veratridine, probably by modulating sodium

channels. HaTX did not show any significant toxic effect on cell viability (without

ouabain/veratridine treatment) even at the highest concentration tested (data not

shown).

HaTx induces a fast neuronal network silencing

The frequency of cytosolic Ca2+ transients are a classical indicator for neuronal network

activity. Using a Ca2+-sensitive dye, we evaluated the effect of HaTX on the frequency of

calcium transients in hippocampal neurons. As shown in Figure 3A, HaTX (0.5% v/v)

induced a fast blockade of Ca2+ transients, since a significant decrease in frequency was

observed in hippocampal neurons after 1 min of HaTX (0.5%v/v) exposure, and a maximal

decrease was observed after 5 min of treatment (Control: 102 ± 7%, HaTX (1min): 31 ±

9%, HaTX (5 min): 4 ± 2 %). The frequency of Ca2+ transients were fast and completely

recovered after 3 min of washout (Figure 3A, lower panel), and no significant differences

were observed with respect to control condition (Control: 102 ± 7%, Wash (3 min):100 ±

5%). These results were highly similar to the effects observed with the classical sodium

channel blocker tetrodotoxin (TTX, Figure 3B), with the difference being that TTX induced

a stronger blockade at 1 min and a slower recovery (15 min after the wash) to elicit the

control activity (Control: 101 ± 5%, TTX 25 nM (1 min): 6 ± 1%, TTX (5 min): 2 ± 1, Wash

(15 min): 133 ± 7%). These results demonstrate a highly reversible effect of HaTX without

the overexcitation observed with TTX withdrawal (Figure 3A-B, lower panels).

On the other hand, we measured the electrophysiological activity of the neuronal network.

The total synaptic activity showed a strong and reversible inhibition when hippocampal

neurons were perfused with HaTX (0.5%, 30 s, Figure 4A). This inhibition was represented

by a reduction in spontaneous current frequency (Control: 16.2 ± 2.9 Hz, HaTX 0.5%v/v:

7.1 ± 1.7 Hz, Figure 4B) and cumulative probability of the current amplitude (Figure 4C).

The electrophysiological recordings correlate with the effects observed on the activity of

Ca2+ transients in the presence of HaTX, similar to TTX blockade of total synaptic currents

(data not shown). Additionally, we measured the effects of HaTX on miniature postsynaptic

currents (mPSCs) and compare them with the effect observed with TTX (Figure 5A). Both

compounds showed a similar inhibition with no significant differences in frequency (HaTX

0.5%v/v: 6.6 ± 0.9 Hz, TTX 25 nM: 5.5 ± 0.9 Hz, Fig. 5B) or amplitude (HaTX 0.5%v/v:

118.6 ± 2.2 pA, TTX 25 nM: 113.8 ± 2.3 pA, Fig. 5C) of the synaptic events. Taken

together, these results indicate that HaTX could have a TTX-like effect, i.e. blocking Na+

channels.

Synaptic silencing induced by HaTX is strongly linked to a TTX-like synaptic

reorganization

To evaluate if the decrease in synaptic activity induced by HaTX was associated with a

change in the synaptic structure, levels of SV2, a key exocytotic protein commonly used

as presynaptic marker, and levels of PSD95, used as postsynaptic marker, were

measured by immunocytochemistry and Western blot, respectively. The acute treatment of

hippocampal neurons with HaTX (0.5% v/v) showed changes in the distribution and a

significant decrease in the immunoreactivity for SV2 (Fig. 6A) in the primary processes

(Control: 100 ± 8%, HaTX: 46 ± 4%, TTX 35 ± 6%) and soma (Control: 100 ± 19%, HaTX:

19 ± 3%, TTX 35 ± 3%, Figure 6B). However, treatment with HaTX or TTX did not produce

changes in SV2 protein expression levels (SV2 Control: 100 ± 11%, HaTX: 101 ± 19%,

TTX: 97 ± 11%, Figure 6C), suggesting that sodium channel blockade induced a neuronal

network disconnection by re-ordering the synaptic structures. Similarly, immunostaining

experiments with PSD95 (Fig. 7A) showed a significant decrease in the signal in the

processes (Control: 99 ± 9%, HaTX: 40 ± 5%, TTX: 47 ± 6%), whereas in the soma, a non

significant decrease in the immunoreactivity for PSD95 was observed in both treatments

(Control: 100 ± 25%, HaTX: 50 ± 6%, TTX: 67 ± 10%, Figure 7B). Similar to SV2, the

postsynaptic marker PSD95 did not show significant changes in its expression levels

(Control: 100 ± 8%, HaTX: 101 ± 17%, TTX: 117 ± 28%, Figure 7C). These results indicate

that acute exposure (1 h) to HaTX (0.5%v/v) causes a synaptic disconnection, similar to

TTX, suggesting that the acute neurotoxicity induced by HaTx involves a synaptic

disorganization that affects the synaptic strength which is dependent on activity (Zhang,

2011).

DISCUSSION

In the present work, we did a functional characterization of an extract obtained from H.

akashiwo (HaTX) and we made a pharmacological comparison to the effects observed

with TTX using in vivo and in vitro models. Currently, the ictiotoxic mechanism for H.

akashiwo remains unclear, although some hypotheses have been suggested. Diverse

studies have shown that H. akashiwo is able to interfere with the natatorium abilities of

species like A. salina (Yan et al., 2004) and correlate this effect with the production of

glycocalyx analogs such as polysaccharide-protein complexes (APPCs) (Yamasaki et al.,

2009) that induce alterations in the nutrition, food intake, development, reproduction and

survival of aquatic organisms (Wang et al., 2006; Xie et al., 2008; Yu et al., 2010).

However, our results suggest a different mechanism of action since the same inhibitory

effect on the natatorium abilities were observed with H. akashiwo, as well as with the

extract (HaTX, Figure 1), discarding any effect associated to the in vivo production of

glycocalyx analogs. On the other hand, H. akashiwo has been classically associated to the

production of neurotoxins similar to brevetoxins (Khan et al., 1997), long lipophilic

polyether chains (10-11 rings) characterized by their excitotoxic properties that are related

with the opening of voltage dependent sodium channels (Aguayo et al., 2006; Dechraoui et

al., 1999; Lombet et al., 1987). However, under our experimental conditions, HaTX

exhibited more of an inhibitory rather than excitatory effect as demonstrated by our data

with A. salina and N2A cells using the neuroblastoma Neuro2a cytotoxicity assay (Manger

et al., 1993); Figure 2). These results suggest the presence of paralyzing compounds in

HaTX, with a saxitoxin- or tetrodotoxin- like behavior, that have not been reported yet in

the literature and that could be associated with the blocking or inhibiting properties

observed in our experiments.

Using neuronal models, we wanted to compare the effects of HaTX with the most classical

voltage dependent sodium channel blocker, TTX. The experiments done on hippocampal

neurons showed a similar inhibition of HaTX as compared with TTX, in terms of its

potency, efficacy to block the synaptic activity, and more interesting, a faster recovery after

toxin withdrawal, suggesting the possibility that HaTX has a more reversible interaction

with voltage dependent sodium channels. In parallel, the immunocytochemistry results

showed that HaTX induced synaptic disconnection to the same extent as TTX, suggesting

that the acute neurotoxicity induced by both toxins involves a synaptic disorganization that

affects synaptic activities and neurotransmission (Zhang and Lisman, 2011). Taken

together, this work suggests that H. akashiwo is capable of producing compounds that can

inhibit neuronal VDSCs, like TTX, and are more closely related to brevenals, classical

sodium channel blockers (Aguayo et al., 2006).

REFERENCES

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FIGURE LEGENDS Figure 1. Heterosigma akashiwo microalgae and Heterosigma akashiwo toxin

extract (HaTX) decrease the mobility of Artemia salina in a concentration-dependent

manner. A) Artemia salina were exposed for 24 h to different algae concentrations (4000 -

75000 cells/mL) in the exponential growth phase. The results are expressed as

percentage of reduction of the control mobility of individual species without algae (n=80).

B) Artemia salina were exposed for 24 h to HaTX extract at different concentrations (0.1 –

5 %V/V). The results are expressed as percentage of reduction of the control mobility of

individual species without the HaTX extract (n=40).

Figure 2. Dose-response curves for Heterosigma akashiwo toxin extract (HaTX)

using the Neuro-2a cells cytotoxicity assay. Neuro-2a cells were treated with ouabain

(300 μM) and veratridine (30 μM) in the presence of different concentrations of HaTX

extract (0.14 – 4.35 %v/v) for 24 h. The HaTX extract caused a dose-dependent increase

in the viability of Neuro-2a cells. Results are expressed as percentage of cell rescue vs log

HaTX concentration. Values represent the mean of three independent experiments. The

error bars indicate SE.

FIGURE 3. HaTX induces a TTX-like reduction in the frequency of intracellular

calcium transients (A,B) Representative fluorimetric recordings obtained from

hippocampal neurons in control, HaTX (0.5%v/v) and TTX (25 nM) treated conditions, and

after wash. (C,D) Effect of HaTX and TTX on the frequency (% relative to control) of

intracellular Ca2+ transients obtained from n > 5 hippocampal neurons. Bars are mean ±

SEM. ***, p < 0.001 (relative to control); †††, p < 0.001 (relative to wash).

FIGURE 4. HaTX induces a silencing in the synaptic transmission (A) Representative

traces of postsynaptic currents illustrate the effect of perfusing HaTX (0.5%v/v/) on

hippocampal neurons. (B, C) The bars show the effect of treatments on the frequency (Hz)

and the frequency distribution histogram of the recordings shown in “A”. The values are

mean ± SEM obtained from n=8 hippocampal neurons. *, p < 0.05.

Figure 5. HaTX induces a TTX-like decrease in the synaptic transmission. (A)

Representative traces of postsynaptic currents illustrate the effect of perfusing TTX (25nM)

and HaTX (0.5%v/v) on hippocampal neurons. (B, C) The graphs show the effect of

treatments on the frequency (Hz) and amplitude (pA) of the recordings shown in “A”. The

values are mean ± SEM obtained from the indicated number of neurons.

FIGURE 6. Acute treatments with HaTX and TTX decrease the levels of the

presynaptic protein SV2 (A) 3D reconstruction of confocal images obtained from control,

HaTX (1 h; 0.5%v/v) and TTX (1h; 25 nM) treated neurons using antibodies against SV2

(green) and MAP-2 (red). (B) The graphs show the number of SV2 puncta per 20 microns

in the processes and soma for each condition. The bars are mean ± SEM obtained from

three different experiments. **, p < 0.01; ***, p < 0.001. (C) SV2 levels in hippocampal

neurons treated with HaTX (0.5 %v/v; 1 h) and TTX (25 nM; 1 h) quantified by Western

blot. β-actin levels were used as internal controls. The bars are mean ± SEM obtained

from three different experiments.

FIGURE 7. Acute treatments with HaTX and TTX decrease the levels of the

postsynaptic protein PSD95 (A) Confocal images obtained from control, HaTX (1 h;

0.5%v/v) and TTX (1h; 25 nM) treated neurons using antibodies against PSD95 (green)

and MAP-2 (red). (B) The graphs show the number of PSD95 puncta per 20 microns in the

processes and soma for each condition. The bars are mean ± SEM obtained from the

indicated number of neurons. **, p < 0.01; ***, p < 0.001. (C) PSD95 levels in hippocampal

neurons treated with HaTX (0.5 %v/v; 1 h) and TTX (25 nM; 1 h) quantified by Western

blot. β-actin levels were used as internal controls. The bars are mean ± SEM obtained

from three different experiments.

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Figure 6Click here to download high resolution image

Figure 7Click here to download high resolution image

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