Will this paper finally be the one that will save my research project?

After my supervisor has seen the images from my second set of experiments, both of us went into a deep puzzle. He suggested that both of us should look deeper into literature that involved locust immunohistochemistry. One day, he sent me a paper that he thought I should ready and hopefully it help me to get the antibodies into the ganglia. Initially I was very skeptical at this paper because I didn't understand why would my supervisor send me a paper on locusts moulting rather than a paper on locusts' molecular biology or genetics which might actually involve immunostaining.

The paper he sent me involves studying the frontal ganglion (FP) and hormones involved during moulting.  The background for this paper is that unlike vertebrates, invertebrate growth requires molting. Successful moultings are essential for insect survival, which requires highly regulated hormonal and behavior coordination.

Past studies have indicated that at least four hormones, pre-ecdysis triggering hormone (PETH), ecdysis triggering hormone (ETH), eclosion hormone (EH) and crustacean cardioactive peptide (CCAP), are involved in the process of two phases of moulting, pre-ecdysis and ecdysis.

During pre-ecdysis, motor patterns are believed to loosen the old and new cuticles. However, the insect does not extricate itself from its old cuticle until ecdysis. The pre-ecdysis behaviours were triggered by the effects of PETH and ETH acting on the central nervous system (CNS). Centrally released EH is thought then triggers ecdysis behaviour, either directly or followed by a release of CCAP within the CNS in a cGMP-dependent manner. CCAP then triggers the motor activities necessary to complete ecdysial behaviour.

The researchers in this paper have previously described a novel central pattern generator (CPG) network situated in the locust FG, and the motor patterns it generates. In the desert locust, Schistocerca gregaria, FG neurones innervate foregut dilator muscles and play a critical role in the control of foregut motor patterns in
different physiological and behavioural states. The researchers have presented the FG as an important target for chemical modulation.

During moulting, the foregut and FG are involved in air-swallowing behaviour. By filling the gut with air, the larval locust can generate enough internal pressure on the body wall to eventually split open the old cuticle, and to then stretch and shape the new adult cuticle and wings after the old cuticle has been shed at ecdysis. Frontal ganglionectomy abolishes air-swallowing and results in difficulty or failure in eclosion and wing expansion. A role for the FG during moulting has been reported for a number of insect species.

In this study, the researchers used suction electrodes to examine the FG CPG as an unexplored target for ecdysis peptides. They also examined the density of CCAP using immunoblotting, and the location of CCAP and cGMP through immunohistochemistry. In their study, they identified ETH, and PETH as FG bursting frequency promoters, while EH as a FG bursting frequency inhibitor.

Figure A illustrated that ETH significantly increased the burst frequency in locusts in CPG. Figure B illustrated both ETH and PETH can increase locusts' burst frequencies, however, ETH was a significantly potent burst frequency promotor than PETH. Figure C illustrated that after the application of EH, locusts' burst frequency stops, but the inhibition is not permanent.

In addition, the researchers also identified CCAP as a potent modulator of the locust FG motor patterns. With applications of CCAP, frontal connectives (FC) and medial pharyngeal nerve (MPN) showed a significant increase in bursting frequency. The increased bursting frequency diminishes when CCAP is washed away with saline. In semi-intact preparations, CCAP also resulted in air bubbles in the crop, which is presumably the effect of air swallowing. Their results imply a novel role for this peptide in generating air swallowing behaviour during the early stages of ecdysis.

Figure A demonstrated that after CCAP application, there is a significant increase in burst frequency within the FC and MPN. However, the effects of CCAP can be diminished after washing the tissue. Figure B illustrated the air bubbles formed on top of the tissue after CCAP application as the result of air swallowing.

Last but not the less, from immunostaining researchers found that one to three CCAP-immunoreactive neurones in the tritocerebrum that extended through the FC nerve and gave rise to extensive arborization within the neuropil of the FG. Their immunostaning examined the variation in the number of CCAP immunoreactive axons in the FG neuropil at the different stages. In mid-larvae only one to two (mostly one) fibres were stained, whereas at the air-swallowing stage at least three afferent fibres were visible. As ecdysis progressed the number of stained axons then declined. A quantitative analysis concluded that the area of CCAP-immunoreactive neuropil increased during airswallowing and decreased at late ecdysis.

These series of immunostaining illustrates the number of CCAP-immunostaining reactive axons and the size of CCAP-immunostaining reactive neuropil in the the FG. These images demonstrated that the number of CCAP related axons increases in size as moulting occurs, but drops at late ecdysis, same logic applies to the size of CCAP related neuropil.

After all findings on regulators on locusts moulting, the part of this paper that might really help me in my project is found in their immunostaining section, when they stated '... (the tissues) were incubated in 0.5 – 1.0 mg/ml collagenase in PBSTX for 30 – 60 min at room temperature to aid in antibody penetration.' This is exactly what I wanted to read! Since my problem was that my antibodies wasn't entering the cells. This definitely gave me new energy on returning to my experiments again.


However, the part I didn't like in this paper was that many of the factors in the immunostaining had a relative large uncertainty in their incubation time. For example, as mention earlier, the tissues were incubated between 30 to 60 mins in collagenase, which might caused a difference by itself in staining results. Furthermore, this would mean I had to try the difference time frames during different incubation processes too to find an optimal time myself.

Ref:
Zilberstein, Y., Ewer, J., Ayali, A., 2006. Neuromodulation of the locust frontal ganglion during the moult: a novel role for insect ecdysis peptides. The Journal of Experimental Biology. 209:2911-2919.

Trial 2

After I collected my thoughts back together and got over about my experiment did not work, I concluded that the reasons it failed could either be because 1) the antibodies would not penetrate through the sheath, or 2) the antibodies are not binding onto the ATPase (which I doubted and really hoped was not the case, because from the background research that was the only Na+/K+-ATPase antibody other researchers have used on insect Na+/K+-ATPase. Therefore, I didn't have much choice in terms of choosing antibodies). After discussing the problem and possible solutions with my supervisor, he suggested that I could use paraffin and sectioning to mount the tissues in wax and section the ganglia into slices to eliminate the problem created by the sheath. Therefore, if this experiment worked, then we can conclude that the first time my experiment didn't work was because the antibodies weren't able to penetrate the sheath and rule out the possibility that the antibodies are not binding on to the ATPase.

After I have my ganglia sectioned by a lab technician from another department, paraffined and stained, I'm ready for round 2 of confocal. This time the staining worked (the green fluorescence),  indicating that antibodies were able to enter the cell body and stained the ATPase. But as one problem is solved, another problem emerged. Because of the fragility and the size of a ganglion, which is roughly the size of a pen's tip, the ganglia were heavily damaged during the sectioning process and orientated in a random fashion. As a result, we were not able to identify the neurons within each ganglion and to make valid comparison between control and heat-shocked ganglia. Consequently, it appears that paraffin and sectioning also may not be the route to go about my experiment. Now, I will need to come up with some other method that does not involve sectioning but yet still allows the antibodies to penetrate through the sheath...

Before the data collection...

If you are going to ask me to sum up my research project in one sentense, it would probably be something like this: using fluorescence antibody to study whether the location/trafficking of the Na+/K+-ATPases differ between a control locust and a locust that has received a heat-shock pre-treatment in their meta-thoracic ganglia (a pile of neurons enclosed by a layer of sheath). Sounds pretty easy you say? Yeah, that's what I thought too, before I started my experiments...

I've learned couple of things in the past year I've spend on doing my Master’s. For example, your experiment always take much longer than what your expected or as indicated on your protocol. But an even more important lesson I've learned was if this is the first time your lab doing a type of experiment, the chances are, it probably will not work at the first try;

My experiment sounded simple enough, dissect out the ganglia, incubate them with antibodies, and then study them through a confocal microscope; but the reality was far from that...

Trial 1:

Like a kid going to his first day of school, I was very excited about starting on my immunohistochemical experiments. I did my research and came up with a feasible protocol. I followed the protocol word to word, incubating the ganglion in antibodies  (red fluorescence) to stain the ATPases and DAPI  (blue fluorescence) to stain the cellular nuclei. Once the samples were ready, I just could not wait to see what they would look like under the confocal.

But to my least expected, this is what came up under the confocal... At first, I had no idea what was going on. But after my supervisor has looked at the images, we concluded only DAPI was small enough to penetrate the dense shealth that is enclosing the ganglia. Consequently, the sheath blocked out all the antibodies, resulting in only those visible cellular nuclei stained by DAPI. The red that we are seeing in our images were the visible antibodies that were the residues trapped inside of the ganglia trachia.

Well, I guess my first trial didn't work, looks like it's time for me to do more research and back to the drawing board again...