The overall goal of this framework is to identify genes involved in broad-range dietary restriction, quantify the environmental information encoded by their expression levels and understand the underlying coding strategy that links environment to lifespan. This method can help answer key questions in the neurobiology of aging field, such as, which genes convey information from the environment to modulate lifespan. Although this framework can provide the insight into the C Elegan sensory system, it can also be applied more generally to any biological system whose components co-operate, to process environmental information.
The main advantage of this technique, is that it quantifies the environmental information encoded by groups of the neurons and determines the coding strategy employed by neuroselquites to convey this information to downstream targets. After culturing OP50 on MGM plates according to the text protocol, prepare 500 milliliters of LB medium in each of two liter erlenmeyer flasks and autoclave them. Inoculate each flask with a single colony of OP50 and grow the cultures at 37 degree celsius and 200 RPM for around 14 hours.
Next, supplement the cultures with 50 micrograms per milliliter of streptomycin. Then shake the flasks for an additional 30 minutes at 37 degrees celsius, before placing the flasks on ice for 15 minutes. Transfer 450 milliliters of each culture into separate 500 milliliter sterile centrifuge bottles, retain the leftover culture on ice, as it will be used to determine the concentration of the bacteria.
Spin down the bottles at 4500 x G and four degree celsius for 25 minutes, then discard the supernatant and store the bottles on ice. With 900 microliters of sterile LB, dilute 100 microliters of leftover culture from each flask. Use one milliliter of sterile LB to zero the spectrophotometer and then determine the OD600 of the 10-fold dilution of each culture.
If for example, the OD600 of the 10-fold dilution of the overnight culture is 0.28, then the culture had an actual OD600 of 2.8. From this example, to arrive at a working stock solution of 1.12 x 10 to the 10th OP50 cells per milliliter, which equals an OD600 of 56, re-suspend the pellet from the 450 milliliter culture in one 20th of the original volume or 22.5 milliliters of sterile S basal solution or SB, supplemented with 50 micrograms per milliliter of streptomycin. Make all subsequent concentrations used in experiments in SB and strep, from serial dilutions of the working stock, using the dilution factors listed in this table.
After culturing and synchronizing C Elegans reporter strains according to the text protocol, use 15 milliliters of SB to serially wash the worms off the three plates and collect the liquid in a sterile 15 milliliter tube. Allow the L4 larvae to naturally sediment and then aspirate all but about 0.5 milliliters of the liquid. In the wild type reporter strains, this type removes any larvae younger than L4.In mutant backgrounds, this step aids in the removal of any arrested larvae, such as, ours, in the case of deaths of an OK3125.
Next, use nine milliliters of SB to re-suspend the worms. Again, monitor the sedimentation rate of the L4 larvae and then aspirate all but approximately 0.5 milliliters of the supernatant once most of the L4 larvae have pelleted before repeating the process. Add 10 microliters of sterile S basal medium, supplemented with 0.1%pluronic F127 to the liquid containing the L4 larvae.
This acts as a surfactant and prevents the larvae from sticking to the interior surface of the plastic pipette tips. Using a P200 low retention pipette tip, gently re-suspend the larvae, and then aliquot 150 microliters onto three 10 centimeter RNAi plates that are seeded with 5 x 225 microliters of egg five RNAi bacteria. Ensure that the worms are equally distributed across all five bacteria lawns.
Once the liquid has absorbed into the agar, remove any non-L4 larvae from the plates that were not eliminated by the washing procedure, by manually picking them off. Then store the plates at 20 degree celsius for 24 hours. To initiate broad range DR, pick off any young larvae that escaped the prior manual removal step, leaving only the one day old adults on the plates.
For each strain, use 15 milliliters of sterile SB strep, to wash the one day old adults from the three plates, into a 15 milliliter tube. Allow the worms to naturally sediment and then aspirate all but 0.5 milliliters of the supernatant. Re-suspend the worms with 9.5 milliliters of SB strep and repeat the sedimentation and wash steps.
After the final wash and aspiration of all but 0.5 milliliters of the supernatant, add 10 microliters of SB Plu and use a P200 pipette tip to gently re-suspend the larvae. Aliquot 100 microliters of the larvae onto an NSC plate, seeded with 5 x 225 microliters of bacteria, at a concentration of 2 x 10 to the ninth cells per milliliter. Distribute the 100 microliters evenly across all five bacteria lawns.
Under a microscope, estimate the number of animals present on the plate, aiming for between 100 and 150 worms per plate. Determine the volume of liquid required to achieve a worm density within this range and then aliquot it onto two additional plates. Adjust the number of worms on the first plate to also fall into this range.
And then store the plates at 20 degrees celsius for 24 hours. The next day, collect the two day old adults and distribute the worms to new NSC plates seeded with the desire to experimental concentration of food. Once the liquid is absorbed into the agar, shift the plates to the desired experimental temperature for 24 hours.
On the following day, collect the three day old adults and distribute them to fresh NSC plates, seeded with the same experimental concentration of food. Once the liquid is absorbed into the agar, return the plates to the experimental temperature for 48 hours. Finally, use a microfluidic device to image the animals before assembling and quantifying the data according to the text protocol.
As shown here, the mean lifespan of the wild type n2 strain, displays a complex response to broad range DR.The magnitude of this response is attenuated in an all mutant of the daf-7 gene, suggesting that this gene affects the ability of the worm to correctly respond to changes in food abundance. The mean expression levels of a transcriptional reporter for the daf-7 gene and the wild type background, also displays a complex non-monotonic response to broad range DR.In the daf-7(genetic background, the expression of this transcriptional reporter is highly attenuated and shows little response to changes in food level. This figure illustrates the estimation of the joint distribution of TPH1 expression in ADF neurons and daf-7 expression in ASI neurons for a given food level.
These bar graphs present the food information encoded, either combinatorially or individually, by ADF, ASI and NSM neurons, based on the gene expression levels of TPH1 and daf-7. Redundant and synergistic characters of the encoding, results from the comparison between combinatorial and individual information encoded by neurons represented by the difference in height of the stacked bars on the right and the information encoded by the full circuit. While attempting this procedure, it is important to remember that replication of information theory to quantify the coding strategy requires an accurate estimation of gene expression distributions.
For this reason, the sample size is a critical factor for the general applicability of this framework. After watching this video, you should have a good understanding of how to perform broad range dietary restriction experiments to identify and characterize new genes that link food abundance to lifespan.
