Seizure 19 (2010) 439–442

Short communication

Baccoside A suppresses epileptic-like seizure/convulsion in Caenorhabditis elegans

Rakesh Pandey a, Shipra Gupta a, Sudeep Tandon a, Olaf Wolkenhauer b, Julio Vera b,Shailendra K. Gupta b,c,*a Central Institute of Medicinal & Aromatic Plants (CSIR), Lucknow 226015, Indiab System Biology & Bioinformatics, University of Rostock, 18051 Rostock, Germanyc Indian Institute of Toxicology Research (CSIR), Lucknow 226001, India

A R T I C L E I N F O

Article history:

Received 5 January 2010

Received in revised form 25 May 2010

Accepted 4 June 2010

Keywords:

Epileptic-like seizure

Convulsion

Ca2+ oscillations

T-type calcium channel

Baccoside A

Caenorhabditis elegans

A B S T R A C T

The 1 mm long Caenorhabditis elegans is one of the prime research tools to study different human

neurodegenerative diseases. We have considered the case in which increase in the surrounding

temperature of this multicellular model leads to abnormal bursts of neuronal cells that can be linked to

seizure or convulsion. The induction of such seizure/convulsion mechanism was done by gradually

increasing the temperature with 1� buffer (100 mM NaCl, 50 mM MgCl2) in adult C. elegans. In the

present experiment it is demonstrated that Baccoside A can significantly reduce the seizure/convulsion

in C. elegans at higher temperatures (26–28 � 1 8C). Furthermore, in T-type Ca2+ channel cca-1 mutant

worms, no convulsion was recorded. Our experimental results suggest that plant molecules from Bacopa

monnieri may be useful in suppressing the seizure/convulsion in worms.

� 2010 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

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1. Introduction

Caenorhabditis elegans offers unique opportunity to study seizure/convulsion related queries. Its short reproductive life cycle (2.5–4days at normal temperature) and 18–20 days life span facilitatedifferent types of investigation related to convulsion by interferencewith drug molecules. Bacopa monnieri (Brahmi) is used in Ayurvedafor improvement of intelligence, memory, and revitalization ofsensory organs.1,2 The nootropic activity of this plant extract hasbeen attributed to the presence of two saponins, namely Baccoside Aand Baccoside B, of which the former is the more important one.3–5

Baccoside A is a triterpenoid saponin (Fig. 1) with molecular weight768.98, topological polar surface area 215.8 A2 and octanol/waterpartition coefficient value 2.53. Different triterpenoid saponins fromB. monnieri have been experimentally verified for antioxidative,6

anticancer,7 antidepressant8 and anticonvulsive9 activities. Bacco-side A has shown to inhibit the excitatory neurotransmission byblockade of calcium ion channels.10

The modification in ion channels has been linked to differentdiseases in humans, including epilepsy. Epilepsy is the result ofhyper excitability (firing and burst) of neurons due to the

* Corresponding author at: System Biology & Bioinformatics, University of

Rostock, 18051 Rostock, Germany. Tel.: +49 381 4987575; Indian Institute of

Toxicology Research (CSIR), Post Box - 80, MG Marg, Lucknow 226001 India. Tel.:

+91 522 2284591.

E-mail addresses: shailendra.gupta@uni-rostock.de, skgupta@iitr.res.in

(S.K. Gupta).

1059-1311/$ – see front matter � 2010 British Epilepsy Association. Published by Else

doi:10.1016/j.seizure.2010.06.005

interruption in the flow of ions (Ca++, Na+ and K+) through voltageand ligand-gated ion channels. Voltage gated Ca++ channels plays asignificant role in different biological processes, including neuro-transmitter release, excitation–contraction of muscle and regula-tion of gene expression as well as neuronal migration. In addition,compounds that directly affect either Ca++ channels or proteinsthat modulate their activity are used to treat a number ofneurological pathologies.11 Williams et al. showed convulsion in C.

elegans by inducing mutation in lis-1 allele (pnm-1) and identicalconvulsions were obtained by them in C. elegans mutant defectivein GABA transmission.12 Similarly it was reported that wormsdepleted for LIS1 pathway components (NUD-1, NUD-2, and DHC-1, CDK-5, CDKA-1) showed convulsions following PTZ and RNAitreatment.13 T-type Ca2+ channels are present and involved indifferent types of cellular activities all over the body. Changes in C.

elegans genes can induce convulsive activity marked by repeatedcontractions more than once either in dorsal or ventral direction(Fig. 2). We have also created seizure index parameters to observeconvulsions in the worm.14 The present investigation was designedto study the effect of plant molecules, especially Baccoside A, onthe seizure activity in C. elegans (wild type). The T-type Ca2+

channel cca-1 mutant worms were also taken in account toexamine the role of such ortholog in the sensitivity of seizure.

2. Materials and methods

The C. elegans culture was maintained on modified nematodegrowth medium (NGM) plates (without adding Ca2+) under

vier Ltd. All rights reserved.

[(Fig._1)TD$FIG]

Fig. 1. Structure of Baccoside A, a triterpenoid saponin from Bacopa monnieri

[(Fig._2)TD$FIG]

Fig. 2. Observations of seizures/convulsions in C. elegans. (A) Effect of temperature on the

normal swimming and seizure burst. (D) Seizures/convulsions behaviors with repeated u

R. Pandey et al. / Seizure 19 (2010) 439–442440

standard conditions.15 The wild type (N2) and cca-1 mutant wormswere grown at 25 � 0.4 8C in incubator and has been worked upon inthe present experiment.

The 0.25%, 0.1% and 0.01% concentrations of Baccoside A wereprepared by transferring required solutions from the stock solutionin 10 ml bacteria feeded plates. 10 stage-IV larvae (L4) of C. elegans

were transferred in control, 0.25%, 0.1% and 0.01% platesrespectively. Similarly, 10 L4 of cca-1 mutant worms weretransferred in bacteria feeded plates. The wild type (N2) L4 weretransferred in newly seeded plates and served as control whichwere incubated at 25 � 0.4 8C. There were four replicates for eachtreatment.

The thermal seizure activity was recorded by placing theseworms in a seizure promoting 1� buffer (100 mM NaCl, 50 mMMgCl2) whose temperature was then gradually increased by usinga variable intensity incandescent light source to generate the heat.A thermometer was submerged in the solution to keep acontinuous track of the temperature. Normally these worms swimby alternately contracting their head in the dorsal and ventraldirection. Animals were scored as showing the seizure behaviour

swimming behavior. (B and C) Alternating dorsal and ventral body bends pattern in

nilateral contraction. (E) Continuous convulsions and paralytic body bend patterns.

[(Fig._3)TD$FIG]

Fig. 3. Effect of Baccoside A on seizure sensitivity and paralysis of C. elegans at 26–

28 � 18C. Error bar indicates the standard deviation.

[(Fig._4)TD$FIG]

Fig. 4. Effect of Baccoside A on seizure intensity in C. elegans at 26–28 � 18C. Error

bar indicates the standard deviation.

R. Pandey et al. / Seizure 19 (2010) 439–442 441

when they repetitively contracted more than once in the samedirection (Fig. 2B and E).

Worms were placed in seizure buffer (1�) and subjected togradually increasing thermal stress. In Fig. 2A, a graph depictingthe swimming pattern is shown. Essentially, 10 s of the relevantswimming behavior is graphed with dorsal body bends repre-sented by upward spikes relative to the midline axis when theanimal is completely straight and ventral body bends representedby downward spikes. With paradigm normal swimming is seen asalternating peaks of ventral and dorsal body bends (Fig. 2C), whileseizure events appeared as unidirectional body bends (Fig. 2D).

The seizure index was recorded by placing the worms in aseizure promoting 1� buffer (100 mM NaCl, 50 mM MgCl2) thengradually increasing the temperature (26–28 � 1 8C) of the bufferusing a variable intensity incandescent light source to generate theheat. The seizures were calculated as on 0–3 scales where 0 = noseizure or convulsion, 1(+) = two twitches in 10 s, 2(++) = two to fivetwitches in 10 s, 3(+++) = more than five twitches in 10 s orcontinuous twitching.

The increment in temperature becomes one of the importantstress factors for induction of seizure or convulsion activity inanimal models. In order to establish the seizure activity in C.

elegans synchronous adult population of wild type worm (N2) wereplaced in isotonic seizure promoting 1� buffer solution (100 mMNaCl, 50 mM MgCl2).

3. Results

In the initial studies, the concentrations of 5%, 1%, and 0.5% ofBaccoside A were found toxic to C. elegans (results not shown)therefore present study was carried out only at 0.1%, 0.25% and0.01% concentration of Baccoside A. The seizure/convulsionfrequency was significantly decreased in C. elegans at 26–28 � 1 8C when compared to control and T-type Ca2+ channel (cca-

1) mutant worms (Figs. 3 and 4). As there was no seizure/convulsionin T-type Ca2+ channel (cca-1) mutant worms, it is suggested that theBaccoside A might be interacting with the CCA-1 channel protein inwild type, resulting in decreased seizure/convulsion.

4. Discussion

In this seizure study, we have shown that T-type Ca 2+ channelaffects the seizure sensitivity in worms, as the cca-1 mutant wormsdid not show the seizure at adult stage. But the wild type wormsshowed seizure intensity at the temperature 26–28 � 1 8C and thereduction in seizure frequency was observed in Baccoside A treatedworms (Figs. 3 and 4) which might be due to the modulation of calciumentry in the cells in C. elegans. T-type calcium channels (CCA-1) plays acritical role in neuronal cellular excitability and have been identified inthe present study as the key factor in epilepsy or convulsion.

For further work, we will construct the 3D structure of T-typeCa2+ channel a-subunits based on homologous mammalian

structures along with computational analysis of binding modeand key molecular interactions between Baccoside A with ionchannels. To further complement our analysis, we will develop apredictive mathematical model accounting for the dynamics ofcalcium channel firing in C. elegans under epileptic-like convulsionconditions and the regulatory effects of ion channel inhibitors likeBaccoside A. Precisely, the model will be based on the extension ofthe previous models derived in order to investigate the regulationof intracellular Ca2+ oscillations16,17 and adapt them to analyse themolecular mechanism along with dose-dependence in Baccoside-mediated regulation of T-type Ca2+ channel firing under epilepticconditions. We also want to investigate the link between calciumchannel dynamics and critical signaling processes controlling themotor circuit in C. elegans (see Valeyev et al.18 for a similarmodeling approach in an analogous calcium signaling systems).Furthermore, our modeling efforts will focus on developing apredictive qualitative modeling linking the timing of ion channelfiring with the frequency and regularity in the movement of C.

elegans, for which we want to pursue a strategy similar to the oneused to link intracellular signaling events and flagella movement inbacterial chemotaxis.19 The approach combining structural biologyand mathematical modeling has also been successfully used by fewgroups in recent times to investigate other biochemical systemsrelated to calcium signaling.20,21

Conflict of interest

None.

Acknowledgments

RP is grateful to Prof. Miguel Estevez, Department of Neurology,School of Medicine, University of Pittsburgh, PA15260, USA, forproviding training to observe the seizure in worms and help todraw Fig. 2. The authors are grateful to the CGC Center,Minneapolis, MN, USA, which is funded by NIH National Centerfor Research Resources, USA for providing the worm culture.

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