The UK Undergraduate Research Program is intended to offer students, particularly in their first and second years, the opportunity to begin to engage in research and scholarship with a faculty mentor. Students in this program may enroll in a special research methods course designed to provide them with practical research and scholarship knowledge, such as how and where to seek funding, how grants are administered, using library and Internet resources effectively for research, and writing research and scholarly abstracts and reports. The following abstracts were the final papers submitted by students who took this methods course in the Spring of 2004 and reported on their on-going research.
The Neural Effects of CO2 in Drosophila Larvae
Nicolas Badre, Research Assistant to Dr. Robin L. Cooper Introduction and Background
Carbon dioxide (CO2) is commonly used as an anesthetic for adult Drosophila melanogaster; however, the mechanism of its actions is unknown. This mechanism is important because it could possibly lead to the discovery of new types of insecticides with the potential to be innocuous to plants and plant eaters. Because mosquitoes have been shown to have sensory structures that detect CO2, we postulated that Drosophila must also contain similar types of receptors, because they share the same kind of environment. Larval insects have never been examined for CO2 sensory neurons. Previous experiments supposed that carbon dioxide affected larvae in the same way that it affects humans: an increase in body fluid acidity causing different behaviors, including anesthesia (Biston and Sillans, 1979). Those experiments also showed that CO2 had different effects than hypoxia, because a high concentration of CO2 and oxygen could also cause anesthesia (Sillians et al., 1969). However, the objective of this current research is to find sensory neurons on the larvae capable of detecting the CO2.Methodology
We tested Canton S, the common “wild-type” laboratory strain of Drosophila melanogaster. This experiment focused on larvae at the beginning of the “wandering” phase of the third instar. Many of the techniques used in this experiment were already used by Cooper and Neckameyer (1999). Each larva was in a sealed agar plate with CO2 injected into the container. We worked in two phases.
Phase 1 – Proving the presence of receptors Body wall movements (bwm) & Heart Beats (HB)
In phase 1, we injected CO2 into the sealed container for a period of 10 minutes, after which the container was opened. We recorded the bwm for the first and last two minutes. If at any time bwm or the HB stopped, the time would be recorded. If the HB stopped, the time when the HB started again, once the container was open, would be recorded. The objective of this test was to quantify the difference between CO2 and hypoxia in the larvae, using common features of the animal.The reaction of the larvae to the CO2
In our effort to identify particular characteristics of the larval response to CO2, we coined several terms to quantify those responses. Shell position designates larvae that are in a curved position. Elongated position designates larvae that are flaccid and look longer than usual. Contracted position designates larvae that have returned to their normal shape after being in elongated position. The responses were tested by placing the larvae under anesthesia for approximately 5 minutes and recording the different behaviors of the larvae during the first minutes and the minutes following the end of the CO2 injection. The objective of this test was to understand and detail the reaction of the larvae to CO2.We repeated the experiment with N2 to make sure that the results were specific to CO2. We also had a control, recording the natural bwm and HB of the larvae without the injection of any gas.
Phase 2 – Finding the receptors
The same flies and methods were used to take care of the larvae at this level, but sealed plates were no longer used. The larvae were placed on tape so that their movements were limited, and a needle was used to aim the flow of the CO2. The CO2 was projected at high pressure in order to prevent rapid diffusion, to enable the analysis of a particular section of the animal. We repeated the experiment with N2 to make sure that the results were specific to CO2. For this experiment, the time at which the heart beat stopped was not recorded, because the time to set up the experiment was rather long and adjustments were sometimes necessary. The animal was divided into two targeted regions: the head and the tail (with the spiracles). The needle was placed accordingly without touching the larva. The aimed flow test was performed on five larvae for each gas.Results
We have shown that larval Drosophila respond rapidly to CO2 (<1 min) by freezing their body movements and contracting the spiracles (respiratory structures). Larvae exposure to 100% N2 gas results in a gradual slowing down of body movement over a longer period of time as compared to the CO2, and does not produce a closing off of the spiracles. Thus, we propose that CO2 receptors drive the central nervous system to initiate particular motor commands that are different than those induced by hypoxia.We have also shown that larval Drosophila only respond to CO2 when it is projected toward the tail region of animal, suggesting that the receptors are located in the tail region.
Future Objectives
Our main future objective is to expand the understanding of the mechanism involved in the neural response to CO2. In order to do so, we will perform a series of neurophysiologic experiments to test the response of each nerve and receptor to CO2. I am currently learning neurophysiologic setups that were recently created to allow the dissection of the larvae without cutting the respiratory structures.Works Cited
Biston J. and Sillans D. (1979) “Studies on the anesthetic
mechanism of carbon dioxide by using
Bombyx mori larvae.” Biochimie 61, No. 2:153- 156.
Cooper, R. L. and Neckameyer, W. S. (1999) “Dopaminergic
neuromodulation of motor neuron
activity and neuromuscular function in Drosophilamelanogaster.”
Comp Biochem Physiol [B]
122:199-210.
Sillans D., Esteve, J., and Legay, J. M. (1969) C.R.
Acad. Sci., 269: 1209-1212.
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