The aim of this procedure is to analyze higher brain functions. In a simple model, DFA larva by means of appetite, associative olfactory learning. This is accomplished by first collecting 30, feeding third instar larvae from a food vial.
Next, the larva are trained for five minutes on a Petri dish with fructose as a reward and an odor container on each side that contains one octal as an olfactory stimulus. Then a second training phase of five minutes is carried out on a dish without the fructose award and with odor containers on each side that contain Amal acetate. After three repetitions of the two training phases, the larvae are exposed to odor containers on opposing sides for a five minute test without a reward.
Ultimately, the relative odor preference of one octal against Amal acetate indicates that drosophila larvae can form an association between olfactory stimuli and sugar reward, and this can be used to study associative learning in a genetically pliable system. This method in combination with genetic interference of neural function can help answer key questions in the learning and memory field, such as what are the neurons that signal reward, or what are the neurons that are necessary or sufficient for the acquisition or the retrieval of a repetitive memory? Demonstrating the procedure will be anti a postop.
A doctoral student from my lab First make 2.5%aros Petri dishes without fructose. To prepare the fructose dishes, add the fructose to the dissolved aros and then pour it into the plate. Next, label each of these dishes with a different symbol that will blind the experimenter.
The fructose containing dish is the conditioning reward, and the plain dish is the non reward. For the condition stimuli prepare a total of three customized Teflon odor containers with 10 microliters of pure one octal. The lids have small holes to allow for evaporation of the odor.
Second, prepare another three odor containers with 10 microliters of amyl acetate diluted one to 50 in paraffin oil. This dilution is as attractive as the 10 microliters of pure one octal begin by collecting 30, feeding, not wandering. Third in from a food vial.
This protocol can be run us any time of day. It is independent of circadian rhythms. Transfer the lava to a Petri dish that contains a few drops of tap water and carefully move them forwards and backwards with a brush.
Then transfer the lava to a second Petri dish that also contains some drops of tap water there. Inspect the larvae for food paste on their body walls. Now place the Petri dish in the experimental setup to train the larvae to associate odors with a particular sugar queue.
Put an odor container on the left side and the right side of one of the two types of dish the stimuli used. Here is one octal, but the choice is arbitrary as the experimenter is blind to the reward. Next place the group of thi larvae onto the middle of the dish.
Close the lid and wait for five minutes while the animals are exposed to the stimuli. Make sure that the larvae can overcome the surface tension of the water droplet so they're not trapped and can explore.Freely. Put a new container of Amal acetate onto a fresh Petri dish.
After five minutes, use a moist and brush to transfer the lava to the opposite type of dish with the opposite stimuli located on its left and right side. So the odor here is amal acetate. Once the larvae transferred, close the lid and allow the animals to explore for five minutes.
When this time expires, the first round of training has been completed. Execute two more rounds of training in the same manner as the first. Transferring the animals by a moist and brush and exposing them to each stimuli and reward combination three times in total.
For the experimental test place, fresh odor containers, one of each stimuli on opposing sites of an uncoated agros only Petri dish. Transfer the trained animals to the middle of the dish. Close the lid and wait five minutes.
After five minutes, use a template under the dish that clearly defines three zones of the dish left, right, a middle count the number of larvae in each zone. The middle zone is a score of non preference and makes the analysis more restrictive and less subject to bias. Finally, to complete the experiment, select 30 new third in staff feeding lava and repeat the training and testing with the opposite combination of reward and stimuli.
Continue using the same odor containers. Several control tests are required to determine whether transgenic animals have normal odorant processing for the reward stimuli. The test for naive odor preference begins with collecting and washing 30 larvae as done for the training and testing.
Next, load the larvae onto an agro rose Petri dish with just one stimuli vial on one side. Close the lid and wait for five minutes. Subsequently, using the template, count the number of larvae on the near side, in the middle, and on the far side of the dish relative to the stimuli.
Perform this test for each stimulus used in the experiment to test for naive sugar preference. Prepare half, half Petri dishes by cutting out half the agros of a cord dish and filling the vacant space with molten agros of the other type. Be careful that both halves match in volume and do not form a defined edge.
This may require practice with the half half dish prepared and at room temperature, placed 30 naive, cleaned off feeding. Third in star larvae at the center of the dish. Close the lid and wait for five minutes.
Subsequently, using the template, count the number of larvae on either side and count those in the middle. The described procedure was used to train and test trenchgenic lava. Using the example stimuli, data was tallied as the number of larvae in different regions of the Petri dishes.
After five minutes out of this data preference, indices were calculated and performance indexes were derived from the reciprocally trained groups. Because the lava tested were transgenic, a choice between a filled odor container and air was also offered as well as a choice between pure aros and aros plus a sugar. An examination of the calculated indexes in box plot revealed that the animals were able to learn to distinguish one octal from ML acetate using fructose agros as an appetite of reinforcer.
The animals recognized both stimuli as attractants and showed a naive preference for fructose aros overplaying aero. This experimental paradigm has been used successfully with a wide variety of olfactory and visual stimuli, as well as a wide variety of rewarding and punishing stimuli. This paradigm combined with the many powerful genetic manipulations available in drosophila is particularly useful in dissecting neural networks, for example, by genetically manipulating specific sets of neurons, defined by gal four expression patterns, the functional roles of those neurons in appetite learning can be assessed.
Following this procedure, along with genetic interference of neuronal function can be used to analyze the neural networks of the larval brain involved in learning and memory. Due to the simplicity of the larval brain, this can be achieved up to the single cell level.
