Seizure: European Journal of Epilepsy
Volume 19, Issue 7 , Pages 439-442, September 2010

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

  • Rakesh Pandey

      Affiliations

    • Central Institute of Medicinal & Aromatic Plants (CSIR), Lucknow 226015, India
    • ,
  • Shipra Gupta

      Affiliations

    • Central Institute of Medicinal & Aromatic Plants (CSIR), Lucknow 226015, India
    • ,
  • Sudeep Tandon

      Affiliations

    • Central Institute of Medicinal & Aromatic Plants (CSIR), Lucknow 226015, India
    • ,
  • Olaf Wolkenhauer

      Affiliations

    • System Biology & Bioinformatics, University of Rostock, 18051 Rostock, Germany
    • ,
  • Julio Vera

      Affiliations

    • System Biology & Bioinformatics, University of Rostock, 18051 Rostock, Germany
    • ,
  • Shailendra K. Gupta

      Affiliations

    • System Biology & Bioinformatics, University of Rostock, 18051 Rostock, Germany
    • Indian Institute of Toxicology Research (CSIR), Lucknow 226001, India
    • Corresponding Author InformationCorresponding 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.

Received 5 January 2010; received in revised form 25 May 2010; accepted 4 June 2010. published online 05 July 2010.

Article Outline

Abstract 

The 1mm 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 (100mM NaCl, 50mM 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°C). 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.

Keywords: Epileptic-like seizure, Convulsion, Ca2+ oscillations, T-type calcium channel, Baccoside A, Caenorhabditis elegans

 

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1. Introduction 

Caenorhabditis elegans offers unique opportunity to study seizure/convulsion related queries. Its short reproductive life cycle (2.5–4 days at normal temperature) and 18–20 days life span facilitate different types of investigation related to convulsion by interference with drug molecules. Bacopa monnieri (Brahmi) is used in Ayurveda for improvement of intelligence, memory, and revitalization of sensory organs.1, 2 The nootropic activity of this plant extract has been attributed to the presence of two saponins, namely Baccoside A and Baccoside B, of which the former is the more important one.3, 4, 5 Baccoside A is a triterpenoid saponin (Fig. 1) with molecular weight 768.98, topological polar surface area 215.8Å2 and octanol/water partition coefficient value 2.53. Different triterpenoid saponins from B. monnieri have been experimentally verified for antioxidative,6 anticancer,7 antidepressant8 and anticonvulsive9 activities. Baccoside A has shown to inhibit the excitatory neurotransmission by blockade of calcium ion channels.10

The modification in ion channels has been linked to different diseases in humans, including epilepsy. Epilepsy is the result of hyper excitability (firing and burst) of neurons due to the interruption in the flow of ions (Ca++, Na+ and K+) through voltage and ligand-gated ion channels. Voltage gated Ca++ channels plays a significant role in different biological processes, including neurotransmitter release, excitation–contraction of muscle and regulation of gene expression as well as neuronal migration. In addition, compounds that directly affect either Ca++ channels or proteins that modulate their activity are used to treat a number of neurological pathologies.11 Williams et al. showed convulsion in C. elegans by inducing mutation in lis-1 allele (pnm-1) and identical convulsions were obtained by them in C. elegans mutant defective in GABA transmission.12 Similarly it was reported that worms depleted for LIS1 pathway components (NUD-1, NUD-2, and DHC-1, CDK-5, CDKA-1) showed convulsions following PTZ and RNAi treatment.13 T-type Ca2+ channels are present and involved in different types of cellular activities all over the body. Changes in C. elegans genes can induce convulsive activity marked by repeated contractions more than once either in dorsal or ventral direction (Fig. 2). We have also created seizure index parameters to observe convulsions in the worm.14 The present investigation was designed to study the effect of plant molecules, especially Baccoside A, on the seizure activity in C. elegans (wild type). The T-type Ca2+ channel cca-1 mutant worms were also taken in account to examine the role of such ortholog in the sensitivity of seizure.

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  • Fig. 2. 

    Observations of seizures/convulsions in C. elegans. (A) Effect of temperature on the swimming behavior. (B and C) Alternating dorsal and ventral body bends pattern in normal swimming and seizure burst. (D) Seizures/convulsions behaviors with repeated unilateral contraction. (E) Continuous convulsions and paralytic body bend patterns.

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2. Materials and methods 

The C. elegans culture was maintained on modified nematode growth medium (NGM) plates (without adding Ca2+) under standard conditions.15 The wild type (N2) and cca-1 mutant worms were grown at 25±0.4°C in incubator and has been worked upon in the present experiment.

The 0.25%, 0.1% and 0.01% concentrations of Baccoside A were prepared by transferring required solutions from the stock solution in 10ml bacteria feeded plates. 10 stage-IV larvae (L4) of C. elegans were transferred in control, 0.25%, 0.1% and 0.01% plates respectively. Similarly, 10 L4 of cca-1 mutant worms were transferred in bacteria feeded plates. The wild type (N2) L4 were transferred in newly seeded plates and served as control which were incubated at 25±0.4°C. There were four replicates for each treatment.

The thermal seizure activity was recorded by placing these worms in a seizure promoting 1× buffer (100mM NaCl, 50mM MgCl2) whose temperature was then gradually increased by using a variable intensity incandescent light source to generate the heat. A thermometer was submerged in the solution to keep a continuous track of the temperature. Normally these worms swim by alternately contracting their head in the dorsal and ventral direction. Animals were scored as showing the seizure behaviour when they repetitively contracted more than once in the same direction (Fig. 2B and E).

Worms were placed in seizure buffer (1×) and subjected to gradually increasing thermal stress. In Fig. 2A, a graph depicting the swimming pattern is shown. Essentially, 10s of the relevant swimming behavior is graphed with dorsal body bends represented by upward spikes relative to the midline axis when the animal is completely straight and ventral body bends represented by downward spikes. With paradigm normal swimming is seen as alternating peaks of ventral and dorsal body bends (Fig. 2C), while seizure events appeared as unidirectional body bends (Fig. 2D).

The seizure index was recorded by placing the worms in a seizure promoting 1× buffer (100mM NaCl, 50mM MgCl2) then gradually increasing the temperature (26–28±1°C) of the buffer using a variable intensity incandescent light source to generate the heat. The seizures were calculated as on 0–3 scales where 0=no seizure or convulsion, 1(+)=two twitches in 10s, 2(++)=two to five twitches in 10s, 3(+++)=more than five twitches in 10s or continuous twitching.

The increment in temperature becomes one of the important stress factors for induction of seizure or convulsion activity in animal models. In order to establish the seizure activity in C. elegans synchronous adult population of wild type worm (N2) were placed in isotonic seizure promoting 1× buffer solution (100mM NaCl, 50mM MgCl2).

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3. Results 

In the initial studies, the concentrations of 5%, 1%, and 0.5% of Baccoside A were found toxic to C. elegans (results not shown) therefore present study was carried out only at 0.1%, 0.25% and 0.01% concentration of Baccoside A. The seizure/convulsion frequency was significantly decreased in C. elegans at 26–28±1°C when compared to control and T-type Ca2+ channel (cca-1) mutant worms (Fig. 3, Fig. 4). As there was no seizure/convulsion in T-type Ca2+ channel (cca-1) mutant worms, it is suggested that the Baccoside A might be interacting with the CCA-1 channel protein in wild type, resulting in decreased seizure/convulsion.

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4. Discussion 

In this seizure study, we have shown that T-type Ca 2+ channel affects the seizure sensitivity in worms, as the cca-1 mutant worms did not show the seizure at adult stage. But the wild type worms showed seizure intensity at the temperature 26–28±1°C and the reduction in seizure frequency was observed in Baccoside A treated worms (Fig. 3, Fig. 4) which might be due to the modulation of calcium entry in the cells in C. elegans. T-type calcium channels (CCA-1) plays a critical role in neuronal cellular excitability and have been identified in the present study as the key factor in epilepsy or convulsion.

For further work, we will construct the 3D structure of T-type Ca2+ channel α-subunits based on homologous mammalian structures along with computational analysis of binding mode and key molecular interactions between Baccoside A with ion channels. To further complement our analysis, we will develop a predictive mathematical model accounting for the dynamics of calcium channel firing in C. elegans under epileptic-like convulsion conditions and the regulatory effects of ion channel inhibitors like Baccoside A. Precisely, the model will be based on the extension of the previous models derived in order to investigate the regulation of intracellular Ca2+ oscillations16, 17 and adapt them to analyse the molecular mechanism along with dose-dependence in Baccoside-mediated regulation of T-type Ca2+ channel firing under epileptic conditions. We also want to investigate the link between calcium channel dynamics and critical signaling processes controlling the motor circuit in C. elegans (see Valeyev et al.18 for a similar modeling approach in an analogous calcium signaling systems). Furthermore, our modeling efforts will focus on developing a predictive qualitative modeling linking the timing of ion channel firing with the frequency and regularity in the movement of C. elegans, for which we want to pursue a strategy similar to the one used to link intracellular signaling events and flagella movement in bacterial chemotaxis.19 The approach combining structural biology and mathematical modeling has also been successfully used by few groups in recent times to investigate other biochemical systems related to calcium signaling.20, 21

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Conflict of interest 

None.

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Acknowledgments 

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

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References 

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PII: S1059-1311(10)00119-6

doi:10.1016/j.seizure.2010.06.005

Seizure: European Journal of Epilepsy
Volume 19, Issue 7 , Pages 439-442, September 2010

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