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NEUTOX-899; No of Pages 6

Brief communication

Synaptic effects of low molecular weight components from Chilean BlackWidow spider venom

Jorge Parodi *, Fernando Romero

Laboratorio de Neurociencia-CEBIOR, Departamento de Ciencia Preclinicas, Facultad de Medicina, Universidad de la Frontera, Montevideo 0870, Temuco, Chile

1. Introduction

The Chilean spider Latrodectus mactans pertains to the genusLatrodectus which is found worldwide (Garb et al., 2004) and isknown as the ‘‘Wheat Spider’’ or ‘‘Black Widow’’ and inhabitsvarious regions of Chile. The bite from this spider in humansproduces a systemic effect called latrodectism or systemiclatrodectism and in some cases results in death (Schenone andCorrea, 1985).

Venom collected from Chilean L. mactans in the VIII and IX regionsof Chile was shown to induce a sustained tonic effect in cardiac andsmooth muscle (Romero et al., 2003). In smooth muscle, themechanism of contraction is related to the permeability of Na+ andCa2+ ions which modulate the contractile response (Nouailhetaset al., 1985) that has a fast, phasic component followed by a slowermore sustained tonic component (Shimuta et al., 1982). Our studiesin the deferent vessel of the rat revealed that the effect inducedby the L. mactans venom is partially dependent of adrenergic and

cholinergic mediators (patent pending). These results raise qutions about the mechanism of action of the toxin on smooth musWe postulated ionic mechanism, to explain same of these systeeffects, based in the idea of described effects of low-moleccomponent from Black Widow venom, over muscle and alterathe cellular physiology (Kiyatkin et al., 1992; Grishin et al., 1993has been postulated that potassium currents, for the importancthese currents in the regulation of muscle contraction andparticipation over calcium homeostatic (Sanborn, 2000). A clasynaptic model was affects for a-latrotoxin and high-molecweight peptides (Auger and Marty, 2000) and dependentpotassium channels for normal regulation of synaptic acti(Pan and Stringer, 1997). We tested the effect of Chilean venomhippocampal neuron, and reported the changes in synaptic activfor have synaptic effects under control and postulate, mechanover neuromuscular union. For another way, we choice a bmodel of cell lines, for observation of passive electric propertiemembrane and not showed receptor or increased numberchannels (Varghese et al., 2006). For these reason used HEK 293 cfor the observation of changes in membranes conductance. In

NeuroToxicology xxx (2008) xxx–xxx

A R T I C L E I N F O

Article history:

Received 23 May 2008

Accepted 27 August 2008

Available online xxx

Keywords:

Synapses

Venom

Spider

Toxic

Black Widow

A B S T R A C T

a-Latrotoxin is the principal component of the venom from the euroasiatic Black Widow spider and

been studied for its pharmacological use as a synaptic modulator. Interestingly, smaller molecular we

fractions have been found to be associated with this toxin, but their cellular actions have not been stu

in detail. The venom from the Chilean Black Widow spider (Latrodectus mactans) does not produc

latrotoxin, however it does contain several small polypeptides. We have recently demonstrated cell

effects of these peptides at the synaptic level using whole-cell patch clamp techniques. Purified ven

from the glands of L. mactans was studied in 12 DIV rat hippocampal neuronal cultures. Venom

concentration of 10 nM was able to decrease neuronal conductance thereby increasing memb

resistance. This effect on the passive properties of the neurons induced a change in action poten

kinetics simulating the action of classic potassium channel blockers. These changes produced an incr

in spontaneous synaptic activity in rat hippocampal cultures in the presence of the venom

concentration- and time-dependent manner. These results indicate that venom from Chilean spid

mactans is capable of increasing cell membrane resistance, prolonging the action potential

generating an increase in synaptic activity demonstrating an interesting pharmacological effect of th

low molecular weight fragments.

� 2008 Elsevier Inc. All rights reser

Contents lists available at ScienceDirect

NeuroToxicology

iumof L.

ium

* Corresponding author at: Laboratorio de Neurociencia-CEBIOR, Facultad de

Medicina, Universidad de la Frontera, Montevideo 0870, Temuco, Chile.

Tel.: +56 45 734041.

E-mail address: [email protected] (J. Parodi).

Please cite this article in press as: Parodi J, Romero F. Synaptic effecspider venom, Neurotoxicology (2008), doi:10.1016/j.neuro.2008.0

0161-813X/$ – see front matter � 2008 Elsevier Inc. All rights reserved.

doi:10.1016/j.neuro.2008.08.006

present study was to analyze the possible participation of potassconductance on the systemic effects observed with the venommactans, recording cellular effects associated a changes in potassconductance in neurons and cells lines.

ts of low molecular weight components from Chilean Black Widow8.006

mailto:[email protected]

http://dx.doi.org/10.1016/j.neuro.2008.08.006

http://www.sciencedirect.com/science/journal/0161813X


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aterials and methods

Spider retrieval

emale adult L. mactans spiders from Chile were capturedng the summer months (December 2005 and January 2006)

the area of Alto Bio in the VIII region (7281605100W, 784502400S)reviously described, taking care not to damage breeding zones

ero et al., 2003). The specimens were maintained separate invidual jars for 30 days without food and given only water inr to stimulate the production and concentration of venom inglands.

Venom retrieval

he spiders were immersed in liquid nitrogen and after 1 minsferred to a phosphate saline buffer solution (PBS: NaH2PO4

, Na2HPO4 0.01 M, NaCl 1.35 M, pH 7.4) at 4 8C. The glandse removed and the membrane that binds them to the base wasioned. Each glands with its poison secreting, was placed into a

containing PBS (25 pairs of glands for 100 ml of PBS) andogenized. The homogenate was immediately centrifuged at� g for 15 min and the supernatant was subsequentlyoted, labeled total venom (TV) and frozen at �20 8C. The

ein content of the TV was measured using the Bradfordnique, with small modifications (BioRad Protein Assay).

Neuronal cultures

he animals were treated and handled according to NIHelines (National Institute of Health, USA). Timed (18 days)nant Sprague–Dawley rats were anaesthetized with ether andanized by cervical dislocation. The embryos were quickly

oved, decapitated and the brains placed in cold Hank’stion. Subsequently, the hippocampi were dissected; mechani-

dissociated using trypsin, collagenase and DNase III and theons were then plated at a density of 350,000 cells/ml ontom culture plates containing a monolayer of glia cells. Neurons

e maintained in culture for 12 days in vitro (DIV) at which pointwere used for experiments. The culture media consisted of

MEM, 2 mM glutamine, and 10% fetal bovine serum. The mediareplaced with fresh culture media every 3 days. All drugs,macological compounds and ions used in the differentrimental protocols were diluted in PBS buffer.

Cell cultures

EK Cells 293 (human embryonic kidney cells) are cultivated inEM (Dulbecco’s-modified Eagle medium, Life Technologies,supplemented by 10% of fetal bovine serum (Life Technologiesand streptomicina–penicillin (200 units each, Life Technol-

s Inc). Growth is carried out fewer than 5% CO2 to 37 8C. Theium is changed every 3 days.

Whole-cell patch clamp

he culture media in the plate was replaced with an externaltion containing (in mM): 150 NaCl, 5.4 KCl, 2.0 CaCl2, 1.0l , 10 glucose and 10 HEPES (pH 7.4). The internal solution

scope (Nikon, Eclipse, TE200-u, Japan). The membrane potentialwas adjusted to �60 mV, the current recorded at 50 ms intervalsand filtered at 2 kHz using commercially available software (AxonInstruments, Inc.). Tetrodotoxin (TTX, 100 nM), a Na+ voltage-dependent channel blocker, was added to the external solution toblock excessive synaptic activity evoked by action potentialsthereby allowing the visualization of miniature synaptic currents(mIPSCs and mEPSCs). The patch electrodes were made ofborosilicate (WPI, Sarasota, USA) and prepared using a horizontalpipette puller (Sutter Instruments, USA). The resistance of theelectrodes was less than 4 MV when filled with normal internalsolution.

Stock solutions for assays containing pharmacological com-pounds, drugs or different ligands were prepared weekly indeionized distilled water and kept at 4 8C. For fast application ofdifferent compounds, a system of external tubes (200 mm internaldiameter) situated 50 mm from the cells was used. Electrophysio-logical recordings were done in the presence and absence of differentdrugs, pharmacological compounds and ions. The solutions contain-ing the different compounds flowed continuously from the inside ofthe tubes by gravity and locally bathed the cell under study.Neuronal recordings were done in the presence or absence of TTX(100 nM) depending on whether the desired information was forminiature currents or potential action recording, respectively.

2.6. Data analyse

The recordings were obtained in pClamp 9 from axoninstrument. The graphic was made using Origin 6 or prism 4, forstatistic analyze used Origin 6. The plotted are means � S.E. Forp < 0.05 the means are considered significant.

3. Results

3.1. Total venom alters cell membrane conductance

Previous reports have shown that total venom from the ChileanL. mactans spider is capable of altering normal processes in isolatedorgans (Romero et al., 2003; Romero et al., 2007). These effectshave been reported as secondary to the action of the venom onionic channels and proton pumps such as the sodium/potassiumexchanger (Robello, 1989; Chanturiya and Nikoloshina, 1994). It isnoteworthy that the Chilean species, although having similar toxiceffects, lacks the a-latrotoxin characteristic of the venom. Wedecided to investigate if there was a mechanism which wouldexplain the changes at the isolated organ level that was related tosome of the ionic alterations previously described. Therefore, westudied the effect of extracellular application of TV from L. mactans

in HEK 293 cells. Voltage–current curves for membrane con-ductance were performed, in voltage clamp mode, used adepolarization protocol from �60 to 160 mV on HEK cells in thepresence and absence of TV. Fig. 1 shows that HEK cells incubated30 min with TV present a decrease in the slope, for a conductancegraph (Voltage vs. current plot) indicating an increase inmembrane resistance (the inverses of conductance for this graph)as compared with control. This finding suggests the possibility ofionic channel closure producing an increase in membraneresistance or reduced the conductance (slope of the plot). To findout if this observation was reproducible with other known

J. Parodi, F. Romero / NeuroToxicology xxx (2008) xxx–xxx

2

ained (in mM): 120 KCl, 2.0 MgCl2, 2 ATP-Na2, 10 BAPTA, 0.5, 10 HEPES (pH 7.4). The cells were stabilized at 22 8C forin before starting the experiments. Current changes in theons were detected using the whole-cell patch clampnique (Hamill et al., 1981) with an Axopatch-200B amplifiern Instruments, Inc., Burlingame, CA) and an inverted micro-

ase cite this article in press as: Parodi J, Romero F. Synaptic effecider venom, Neurotoxicology (2008), doi:10.1016/j.neuro.2008.0

molecules, we used the same protocol in cells cultured with TEA(a potassium channel blocker) or with gramicidin, an agent knownto perforate membranes (i.e. increase in conductance). Fig. 1Bdemonstrates that both compounds vary the slope of the curves ascompared to control. Interestingly, the channel blocker TEA had asimilar effect as that observed with TV. The graph in Fig. 1C



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demonstrates the membrane resistance in the absence andpresence of the venom. Cells exposed in acute manner with TVshowed a significant increase in membrane resistance.

3.2. Latrodectus venom alters membrane and action potentials in

hippocampal neurons

The effects of a-latrotoxin in the synapse has been described byseveral investigators; however the mechanism by which thevenom induces changes is still unclear. It is currently accepted thatthe interaction of a-latrotoxin with cell membranes (Krasilnikovand Sabirov, 1992; Orlova et al., 2000; Hlubek et al., 2003) resultsin the formation of a pore permeable to calcium and this is themechanism by which the venom alters synaptic activity (Henkeland Sankaranarayanan, 1999). The presence of other small-molecular weight molecules having a synergistic action have beendescribed (Kiyatkin et al., 1992; Grishin et al., 1993). Our results,however, suggest that the fragments we studied are capable ofaltering the normal functioning of isolated organs and could bemodifying membrane conductance. Fig. 2A shows representativetraces of action potentials from hippocampal neurons in theabsence or presence of TEA and TV. The traces indicate that TEAalters the action potential, lengthening the repolarization phase(Schwartzkroin and Prince, 1980) which is mediated by potassiumchannels (Woodson et al., 1978). The venom is also able to alter theaction potential kinetics in a manner parallel to TEA suggestingsimilar mechanisms. Fig. 2B current clamp traces, acquired in thepresence of TTX (100 nM) for prevented potential action, fromneurons exposed to TEA or TV. As can be seen, TEA produces alengthening on the action potential, a similar effect found with TV.Fig. 2C shows the change in membrane potential, acquired using

3.3. TV increases synaptic transmission in cultured hippocampal

neurons

As previously mentioned, a-latrotoxin is capable of altesynaptic activity, however smaller peptides appear to havcomplementary effect. Our results suggest that in the absence olatrotoxin, such as in our Chilean L. mactans model, the smafragments play an active role in producing the effect seen withvenom. This is in agreement with the systemic effects observed wthe venom from Chilean L. mactans (Romero et al., 2003; Romet al., 2007) as well as with experiments in isolated organs (Romet al., 2003). Fig. 3A shows representative traces of synaptic actiin the absence or presence of TV. Neurons cultured in the presencTV display a significant increase in synaptic events. Interestindenatured venom, boiled for 45 min at 96 8C, has no effect onneurons indicating a specific participation of the peptides presenthe venom (Fig. 3B). Fig. 3C and D reveal a time- and concentratdependent action of TV as reflected in an increase in the frequencevents with longer incubation times and higher concentrationthe venom. Both figure, suggested a pharmacology effects oversynapses, because induced to think a time depend effectsconcentration response, two characteristic of physiological efover receptors or protein. In addition, Fig. 3C demonstratesthese effects are reversible because when the venom is removedsynaptic frequency returns to control levels. On the contrary, higconcentrations of the venom are unable to alter synaptic actisuggesting an antagonistic effect (Fig. 3D), not explored butinteresting in biotechnology application. Therefore, we can sugthat the alteration in synaptic activity in the presence of the venmay be due to blockage of potassium channels, similar tosensible and changes in membrane potential.

Fig. 1. Effects of Chilean Latrodectus mactans venom extract on membrane conductance. The data were obtained from HEK 293 cells. The cultures were exposed to chr

treatment with Chilean Latrodectus mactans venom extract (7.5 mg/ml, 30 min). (A) Shows an I–y relationship in absence or presence of venom. (B) Shows I–y curves

compare the effects of gramicidin (10 mg/ml) and TEA (100 nM) with the venom. (C) Shows values for membrane resistance in the absence and presence of venom. The

are mean � S.E. from 16 different cells (*p < 0.05).


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current clamp, for measured the changes in the potential andreveals that both TEA and TV significantly decrease the membranepotential to values near the action potential threshold. These datademonstrate a change in the passive properties of the membranesin the presence of TV, similar to TEA, indicating the closing ofpotassium channels.


Noteworthy, TV from L. mactans has demonstrated to bpotent stimulator of synaptic activity in cultured hippocamneurons despite the fact that it lacks latrotoxin. Therefore, our dprovides the first evidence that polypeptides present in L. mac

alter synaptic activity in a manner similar to a-latrotoxin, but fdifferent mechanism.



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. Effects of Chilean Latrodectus mactans venom extract on membrane potential from hippocampal neurons. The data were obtained from 12 DIV neurons exposed to acute

cations of Chilean Latrodectus mactans venom extract (7.5 mg/ml). (A) Shows action potential traces, in the absence and presence of TEA (100 nM) or venom (7.5 mg/ml).

ows membrane potential traces, with TTX (100 nM) in absence and presence of venom. (C) Graph showing membrane resistance values in the absence and presence of

m. The bars are mean � S.E. from eight different neurons (*p < 0.05).

. Effects of Chilean Latrodectus mactans venom extract on synaptic activity. The data were obtained from 12 DIV neurons exposed to acute applications of Chilean

dectus mactans venom extract (7.5 mg/ml). (A) Shows synaptic currents obtained in the absence and presence of venom or boiled venom. (B) Shows miniature synaptic

nt frequency in the presence of the venom. (C) Graph of synaptic frequency at different times incubation and (D) data showing the effect of different concentrations of

enom. The bars are mean � S.E. from 19 different neurons (*p < 0.05).


iscussion

revious report, in our laboratory and work for development ant (Under way in countries PCT) was provide informationt the component of the Chilean venom, form L. mactans. We

ase cite this article in press as: Parodi J, Romero F. Synaptic effecider venom, Neurotoxicology (2008), doi:10.1016/j.neuro.2008.0

described a fractionation of the venom by HPLC–MS, and we foundthe absence of the higher weight component, like a-latrotoxin, thistoxin, is the principal component in Black Widow venom andexplain mostly of systemic effect secondary to this venom. A fewreport, do it description of the low weight component, present in



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the venom but not explored. The Chilean venom, not presentshigher component (minor to 10 kDa, data not show) but, theChilean venom can induce similar effects to another Black Widowvenom in absence of a-latrotoxin. Agreement with previousstudies (Romero et al., 2003, 2007), our data show thatpolypeptides present in the venom from Chilean Lactrodectus

mactans spiders increase spontaneous synaptic activity in hippo-campal neurons and changes the passive properties of themembrane, cells lines.

We suggested, potassium current, for the importance of thiscurrent over membrane potential (Gutman et al., 2003) and thecontrol in the synaptic activity (Dodson and Forsythe, 2004; Yuanand Chen, 2006). Work by Grider and Makhlouf (1988) demon-strated that the tonic response in smooth muscle is dependent onthe influx of calcium into the cell. Similarly, synaptic activity isdependent on calcium homeostasis in the presynaptic button(Cousin and Robinson, 2000). Therefore, the neuronal responseobserved in the presence of total venom could be related to calciuminflux as a result of the change in membrane potential, secondaryeffect over calcium, for regulation of potassium channels (Pan andStringer, 1997; Yuan and Chen, 2006) (Fig. 2).

Studies done in isolated organ models determined thatincreased muscle contractility is due to changes in ionic con-ductance, for example potassium current (Doi et al., 2000;Darblade et al., 2006). However, we have not described particularpotassium current. Was explored the evidence of the activationor modulation of channels and the relation with another effectsof the TV. The presences of Kv1.1 in the models used, areimportant suggested, because these channels are blocked by TEAand this channels are important regulator of membrane potential(Gutman et al., 2003). All this is in relation with our data, whenshowed changes in membrane conductance in the presence ofthe venom and TEA (Fig. 1) most likely due to alterations in theTEA sensible conductances (Fig. 2). This finding is importantsince influx of calcium into the synaptic button occurs due to achange in membrane potential that can be regulated bypotassium currents (Charlton et al., 1982; Baba et al., 2003). Inaddition, voltage-dependent calcium channels are sensitive tovariations in voltage resulting in either a higher or lower synapticactivity (Parker, 2000).

However, it is important to recognize that in the classic modelfor the venom from this species of spider, a-latrotoxin isresponsible for depleting synaptic vesicles (Ashton et al., 2001)by disregulating calcium influx and increasing synaptic activity(Scheer et al., 1985; Magazanik et al., 1992) We did not study ifthese effects are reproducible with the venom from L. mactans,however we postulate a new effect of small molecular weightpeptides presents in this venom. We suggested, channelsmodulation for induce changes describes pervious in ourlaboratory and possible to think in a potassium channels, forexplain some of the effects describes or conductances sensible toTEA. More research is needed to study the chronic effects of thesepeptides in order to better understand the systemic effectsproduced by the venom in the spider L. mactans from Chile andfor postulated the channel or channels altered for this venom. Wedo it a fast report for a possible mechanism for this venom andcontinued explored the effects of single peptides, for a biotechnol-ogy development.

Conflict of interest: Patent pending.Funding source: FONDEF-D02I1024 and FONDEF-D05I1041

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ase cite this article in press as: Parodi J, Romero F. Synaptic effects of low molecular weight components from Chilean Black Widowider venom, Neurotoxicology (2008), doi:10.1016/j.neuro.2008.08.006


Technology

Parodi et al 2008 veneno y neuronas