Measuring Glutathione-induced Feeding Response in Hydra

In this video, we describe a method for measuring the feeding behavior in Hydra. In response to reduced form of glutathione, Hydra feeds in a Variety of animals, nematodes, crusta, and newly hatched fishes. On the outer Layer of this two cell layered animal, there are specialized cells called nematocysts.

Nematocysts have triggers, which when activated by the movement of prey in the surrounding fluid causes the discharge of the stringing threads. The stinging threads are inserted into the prey. The injured prey releases glut glucose.

Ion hydro has a chemo sensation for detecting the glutathione in surrounding liquid. This triggers a feeding behavior involving its nervous system. The feeding behavior in Hydra includes curling of the tentacles to bring the prey towards a hyperscope opening of the mouth and engulfing of the prey.

In fact, adding glut ion the surroundings causes the feeding reaction in hydra, even in the absence of prey. To understand the interplay of chemo sensation and nervous system, we need to quantize the behavior and create accurate and dependable measures. In a few earlier reports, the time for which mouth is hydro remains open is taken as a measure of the feeding response, but observing the duration of mouth opening in the hydro polyps is tedious and is subject to variation induced by uncontrollable parameters such as the orientation of the mouth due observations.

Another method of quantification of the behavior is based on the number of hydra in a population showing the feeding response. However, since feeding response is an individual quantitative parameter, the population based approaches are subjective variations caused by the opinion of the individual observer. The main advantages of the alternative method that we describe here is that it's very easy to execute and highly reproducible.

Maintain hydra at 18 degrees Celsius under 12 hour light. 12 hour dark cycles in a medium contain in a glass bowl and feed them daily with emia for measuring the feeding response. Transfer one mature hydro polyp to a well of a 24 Well plate remove residual medium from the well using a pivot.

This is facilitated by tilting the plate, discard the excess medium, and then immediately add 500 microliters of fresh medium Preparation of glutathione Solution. A nine micromolar solution of glutathione is prepared using hydro medium. Remember, glutathione solution is prone to oxidation, so always use freshly prepared glutathione solution.

For each Experiment, transfer the plate to the imaging platform of a microscope. Having provision for image recording use a dark background such that the behavior of hydro polyp can be clearly imaged against the contrasting background. Make sure that the polyp is located along the central part of the well captured an image of hydra in the relaxed state.

This will be a zero time point observation. Quickly add nine micromolar glutathione solution to reach a final concentration of three micromolar in the, well. Start the timer immediately after adding glutathione solution and capture images after every 15 or 30 seconds for four to five minutes without changing the magnification settings.

For measurements, we use gim version two, gim. A GNU Image manipulation program is a software freely downloadable from the internet. Open each of the captured images in gim.

This program has a measure tool available from menu tools. Measure the distance between hypers, stoma, apical end of each of the tentacles can be measured using this tool. This distance is called tentacle spread.

Calculate the average tentacle spread for each polyp before and after glutathione. Calculate the ratio of average tentacle spread at zero time. Point to that at each of the subsequent time points.

Let us call this relative tentacle spread. Do such measurements for at least 20 Polyps for Starvation. Transfer a few hydro polyps to a separate glass bowl and do not feed them for five days.

Feed the control polyps daily with artia on the day of the experiment. Feed the control group of Hydra with artia for one R and use these hydra for the subsequent experiments. Measure the feeding response of the starved hydra in comparison with the hydra from the control group by the method described earlier in this video to avoid any bias due to time of alternate, the measurements of each of the starved and controlled hydro polyps.

Glutathione causes hydra to exhibit curling of the tentacles towards the mouth. Such curling brings apical ends of the tentacles closer to the hypo storm. This results in the reduction in the linear distance between apical ends of the tentacle and hyper storm or tentacle spread.

The relative tentacle spread after adding medium. Lacking glutathione reduces only transiently. It bounces back to one in about a minute.

Starvation is known to cause enhancement of the feeding behavior induced by glutathione. The tentacle spread one minute after adding glutathione was significantly lower for the staff group as compared to the satiated Group. All animals use chemo sensation for detecting food.

Hydra uses a chemical signal in form of glutathione released from the injured prey, Has a simple nervous system without any brain-like structure, so the molecular mechanism of chemo sensation and its feeding status dependent modulation are also likely to be simple. Understanding this mechanism may the shed light on the evolution of a regulation of feeding in higher animals. However, neither the receptor which can bind glutathione nor any other components of the downstream signaling cascade are reported in Haida to this date.

Pharmacological inhibitors of common chemos sensory signaling pathways and the use of advanced molecular biological techniques to alter the expression of such signaling proteins and glutathione receptor can prove to be powerful tools for understanding the molecular mechanism of feeding. In Hydra, however, a reliable measure of altered feeding behavior is needed to understand the extent of any modifications caused by such experimental investigations. Here we have presented a method which is not only reliable, but also easy to perform and is highly reproducible.