Name: measuring glutathione-induced feeding response in hydra
Uploaded: 2014-11-16T15:00:00
Description: Watch this Scientific Journal Video about Measuring Glutathione-induced Feeding Response in Hydra at JoVE.com

Authors are thankful to K. P. Madhu, Nita Beliappa and staff of the Media Centre of Indian Institute of Science Education and Research, Pune for their help in the video production. The work was supported by funding under the Centre of Excellence program of Department of Biotechnology, Government of India to SG and postdoctoral fellowship by Department of Science and Technology, Government of India to RK.

1. Hydra Culture and Measurement of the Feeding Response
<ol>
	<li>Maintain hydra polyps in culture by feeding them daily with artemia and keeping them in a medium (1 mM Tris-HCl buffer, pH 7.6, 1 mM NaCl, 1 mM CaCl2, 0.1 mM KCl, and 0.1 mM MgSO4) contained in a glass bowl at 18 &#176;C under 12 hr light-12 hr dark cycles as described earlier12.</li>
	<li>For measuring the feeding response, transfer one mature hydra polyp having 5 to 6 tentacles to a single well of a 24-well plate. Remo...

<ol>
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Feeding behavior in hydra represents one of the most ancestral chemosensory systems in the metazoans. Although the presence of GSH in the crustacean fluid released after nematocyst-assisted prey capture was detected long ago1, neither the GSHR protein nor the putative encoding gene/s have been characterized from hydra to date. Few attempts have been made to characterize GSH binding proteins in hydra8, 14, 15. However, the identity of these putative receptor proteins remains obscure and very few othe...

Hydra is the most primitive organism possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey1. It feeds on a variety of animals such as nematode, crustacea, insect larvae, tadpoles and newly hatched fish1. The movement of these prey organisms causes mechanosensory discharge of the stinging capsules called nematocysts from hydra, which are inserted into the prey2. GSH present in the fluid released from the injured prey subsequently activates the feeding response in hydra which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth, and consequent engulfing of the prey. Multiple molecules, such as dopamine3, glutamate4, GABA, glycine5, NMDA receptors6, and allatotropin7, have been shown to be involved in the feeding response in hydra. It has also been shown that the chemosensory response induced by GSH is modulated by the feeding status of the animal such that starved hydra exhibited enhanced feeding response1. Such an increase in the GSH sensitivity is biologically relevant since under starvation hydra need to find its prey at higher sensitivity. 
Although the feeding response induced by GSH can be clearly observed under microscope, the methods typically used for measuring the feeding response observations are non-quantitative. In most of the cases, the time during which the mouth of the hydra remains open was taken as a measure of the feeding response8,9; whereas in another case, quantitation was based on the number of hydra out of a population showing the feeding response10. However, observing the mouth opening time of the hydra polyps is cumbersome and subject to variation induced by uncontrollable parameters such as the direction of the mouth orientation during observations. Similarly, since the feeding response is a quantitative parameter, population-based approaches are subject to variations/errors caused by the opinion or observational bias of the individual observer. To circumvent these issues we have developed a method for the relative quantification of the feeding response in hydra (Hydra vulgaris Ind-Pune11) based on the distance of the apical end of the tentacle from the mouth of the hydra polyp.

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			<td height="21" style="height:21px;width:223px;">Microscope</td>
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			<td height="21" style="height:21px;width:223px;">Camera for the microscope</td>
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			<td height="21" style="height:21px;width:223px;">Reduced glutathione</td>
			<td style="width:73px;">Sigma</td>
			<td style="width:123px;">G4251</td>
			<td>Stored at 4&#176;C. Bring the bottle to room temperature before opening to avoid oxidation</td>
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measuring glutathione-induced feeding response in hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Glutathione causes hydra to exhibit curling of the tentacles towards the mouth for the purpose of engulfing the prey. Such curling of tentacles brings apical ends of the tentacles closer to the hypostome. This results in the reduction in the tentacle spread, or the linear distance between apical end of the tentacle and hypostome (Figure 1). The relative tentacle spread, or the ratio of average tentacle spread before and after adding glutathione, averaged across multiple polyps reduces over time. The rela...

Watch this Scientific Journal Video about Measuring Glutathione-induced Feeding Response in Hydra at JoVE.com

Measuring Glutathione-induced Feeding Response in Hydra

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

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