The overall goal of this procedure is to utilize the Drosophila model system to correlate behavioral outputs with changes in neural architecture. This is accomplished by first setting up population cages of adult drosophila to collect larvae. The next step of the procedure is to assess locomotor ability of late second to early third instar larvae.
The third step is to assess the feeding behavior of the larvae by observing the extension and retraction of their mouth hooks. The final step is to dissect and prepare proven ventricular samples from late third instar larvae using immunohistochemical techniques. Ultimately, results can show how aberrations in neural architecture during development relate to behavioral changes through the combined use of behavioral assays and immunofluorescence microscopy.
The implications of this technique extend towards a better understanding of the development of neural architecture, which allows one to better understand the progress of neurological diseases. Demonstrating the procedure will be prag bot a graduate student in my laboratory. Maintain drosophila population cages at 25 degrees Celsius on a 12 hour light dark cycle.
As long as the control and experimental groups are exposed to the same lighting conditions, then this technique can be performed in a standard laboratory setting. Using established populations collect larvae by allowing females to lay eggs overnight on apple juice. Agar plates change the plate in the morning and place a small dollop of yeast in the center of the collected plate.
The yeast will attract the hatched larvae, allow the eggs to develop for 24 hours at 25 degrees Celsius, and on the next day, collect the first instar larvae from the paste. Using a metal spatula, transfer the larvae to a fresh apple juice or grape juice plate. Additional yeast paste can be added to nourish the larvae until the assays are performed to collect second and early third Instar larvae allow the first instar to age for 40 to 48 hours.
During this timeframe, feeding rates are constant to collect. Late third instar larvae raise the larvae in bottles. Raising late third instar larvae on juice plates can be harmful due to agri buildup in the somatic gastric system.
A larvas age can be confirmed by the mouth hook morphology as there are distinct changes in this structure. With each larval mold, collect the late second and early third instar larvae by gently and extensively washing the juice plate with water and pouring the larvae into a mesh filter, then proceed with behavioral analysis. Place a single late second to early third instar larva on a 2%acer substrate in a 100 millimeter tissue culture dish, and allow the larvae to acclimate for 30 seconds for exactly one minute.
Using a counter tally each posterior to anterior movement over the substrate score each contraction that occurs over three quarters of the animal's body. When the larvae sways side to side, but does not actually change position, these movements are not scored. Occasionally a complete anterior to posterior contraction generates a backwards movement like forward locomotion.
Backward locomotion is also scored. Replace the assay plate after 10 animals at most are tested in total. Make at least 20 recordings per experimental condition.
Score each larva used in the locomotor assay in the feeding assay using blunt inox. Number five, tweezers. Carefully transfer a larva from the locomotor agri plate to the center of an agri filled plate overlaid with five milliliters of a homogenous yeast solution.
In the yeast solution, the larvae will largely remain in place and feed feeding correlates directly to the rate of mouth hook contractions, which are seen as the extension and retraction of the cephalon pharyngeal scle rights Allow larvae to acclimate for 30 seconds, and then for one minute, record the number of mouth hook contractions using a counter. Begin by loading freshly prepared 4%EM grade formaldehyde in PBS into a three well spot glass. Then transfer a wandering third instar larva to a dissection dish with PBS.
With one forcep hold the posterior end and with the other hold the mouth hooks while holding the posterior end immobile. Gently pull on the mouth hooks to gain access to the guts. Remove any associated tissues like the salivary glands, brain, fat body, and so forth.
Then transfer each gut to the three well dish containing the formaldehyde fix. Incubate the guts at four degrees Celsius overnight in an opaque tissue culture box. The next day before removing the fixation solution, remove the gastric seca and clip the midgut near the proven ventriculars so that projections can be clearly viewed without impediment.
Now, replace the fixative with one XPBT and allow the tissues to incubate for 10 minutes with gentle rocking. Perform this PBT wash six times. Next, add serotonin, which will enhance serotonin signaling without affecting neural architecture.
Then incubate the guts for an hour at four degrees Celsius without rocking. After an hour, thoroughly wash the guts with PBT six times as done to remove the primary antibody. Then incubate the guts overnight at four degrees Celsius with anti serotonin primary antibody.
The next day, repeat the six PPT washes and follow with the secondary antibody incubation at four degrees Celsius for 90 minutes. Use one to 400 Alexa floor 5 68 goat anti-US, or one to 400 anti rabbit IgG. Remove the secondary antibody using the PBT wash regime and then incubate the guts in four millimolar sodium carbonate with gentle rocking for 10 minutes.
The guts can then be mounted in 4%N propyl gallate with 20 millimolar sodium carbonate as a buffer and viewed under fluorescence at 400 x magnification for analysis. Avoid taking images at the posterior end of the proven ventriculars because these fibers are tightly bundled. More posterior fibers in the midgut are more branched because they fasciculating once they enter the tissue.
Neurite fibers can be quantified by using commercial software like neuro lucita and neuro Explorer by working it out manually or by using the freely available software. Simple nerite tracer images that are not clear will make it difficult to distinguish between the fiber and varicosities and should not be used if the fiber architecture is clearly distinguishable from the background. And if individual varicosities can be identified along the nerite length, the preparation is appropriate for analysis.
Also, if individual varicosities can be identified from the rest of the fiber, this is another indicator of a quality image for analysis. The axonal fibers projecting from the brain are bundled into the recurrence nerve and are inappropriate for analysis until they reach the proven ventricular where they separate and innervate. The steamatic gastric system Analyze all the fibers with the exception of those out of the range of focus.
In some cases, the fibers will curve between multiple planes of focus, trace the individual fiber projections from the brain to the proven ventriculars and count the varicosities and branches, then calculate the frequency of large varicosities per unit length. Studying the serotonergic feeding circuit is effective for analyzing the influence of particular factors on nervous system development. By quantitating feeding rate, it is possible to link the axonal architecture of the feeding circuit with its functional output.
The locomotor assay shows no difference in the locomotor responses between control and mutant genotypes. If the mutations only affect the feeding circuit, the mutant ellipsoid body open. EBO three has a structural defect in the ellipsoid body of the central complex.
The wild type parental strain. CSWU revealed that EO three results in depressed feeding while locomotion is unaffected. The developmental defects in EO three mutants affected the urate architecture of the gut.
These larvae displayed more branching as well as more varicosities, both small and large, along the urate length. Note, the branch nodes at the arrows, the varicosities at the arrow heads, and the large varicosities at the asterisk. These changes in neural branching were quantified for statistical analysis.
When attempting this procedure, it's important to remember not to damage the larvae or the dissected tissue samples.
