The Brain@McGill Prize for 1st place

Sunburns and Pain: Insights from Fruit Flies

Haitian (Billy) He 

Supervisor: Dr. Donald van Meyel

Anyone who has gone through the recovery process from severe physical injury willattest to two facts: despite itsbriefness or lightness, contact with the affected area can be painful and further injury to the area will be excruciatingly painful. There is an analogue to these symptoms for burn victims as well. We sensitize ourselves to lower temperatures and respond more severely to painful heat stimuli. In scientific terms, allodynia is the sensitization to previously non-painful stimuli, and hyperalgesia is the hyper-sensitization to already painful stimuli. The goal of my project is to elucidate the underlying signalling mechanisms that induce allodynia/hyperalgesia. Such knowledge is immensely beneficial for developing treatments for a variety of illnesses and injuries.

One of the biggest issues in patient care is pain reduction. Allodynia and hyperalgesia usually accompany injury as critical defense mechanisms for guarding healing tissues. Under normal conditions, the appearance of both of these phenomena is actually quite useful for expediting the rate of recovery. However, in certain cases, they persist past full recovery, resulting in chronic pain. This persistent hypersensitivity is quite prevalent and leads to a wide variety of disabilities, suffering and costs to society. Current treatments, like opiate administration, often result in tolerance, dependence, nausea, etc. These limitations motivate efforts, similar to my own, to produce more efficient and safe alternative treatments.

My research involves measuring the effects of injury on Drosophila (fruit fly) larvae that we genetically modify. Larvae have a surprisingly robust sense of pain that is easily observable(1). Simply, we give larvae sunburn (controlled UV irradiation) to effect superficial tissue damage. In normal larvae, this generates a typical allodynic/hyperalgesic behavioural response – the larvae actually roll towards the painful stimulus – which we demonstrate using our custom-made heat probe. However, from our own work and others (2-4), we know that alterations to the Hedgehog signalling pathway (a prevalent signalling pathway among all vertebrates, named after the primary secreted protein Hedgehog) can modulate and even abolish this behaviour. By controlling which proteins are affected, I hope to refine or discover new aspects of the signalling system for the benefit of new treatments.

Unfortunately, there is rather large gap in our understanding of the players involved in the Hedgehog pathway in the context of pain transduction. In general, we know that without Hh binding to its receptor during and after the injury, there would be no induction of allodynia/hyperalgesia (In more physical terms, it’s analogous to being able to bump your recently healed broken arm and feeling absolutely normal). Unfortunately, to target and disrupt the Hh gene would be disastrous as it is required in a huge number of roles outside of pain sensitization, resulting in complex and unwanted side-effects. The better alternative is to be able to disrupt signalling specifically in the neurons that transmit the pain.

Through our lab’s previous work, we have shown that two proteins are necessary for active Hh signalling – Interference Hedgehog (Ihog) and Brother of Ihog (Boi) (2). So far, they seem to be doing the exact same role in every tissue system previously investigated and can compensate for one another, yet this theory is untested in the pain sensing neurons of larvae. And so, my project was born. Larvae, either having only functional Ihog or Boi or both (i.e. wild-type, as a control), would be irradiated, causing mild tissue damage in the body wall, putatively sensitizing the pain neurons that go to innervate it. Following irradiation, I would test for the presence and severity of the allodynia/hyperalgesia. Our initial hypothesis was that single mutant larvae would show reduced allodynic/hyperalgesic responses as a result of the loss of one of Hh’s binding partners, Ihog or Boi.

There were two major surprises that came along while recording the responses of the larvae. Firstly, even without UV irradiation, the single mutant larvae actually exhibited a heightening of sensitivity to thermal stimuli (Figure 1B). This runs counter to our expectations of the effects that absence of these proteins would produce. In fact, we see a response analogous to over-activation of the pathway! Moreover, as seen in Figure 1B, though Boi mutants seem to generate some mild hypersensitization, Ihog mutants show dramatically increased baseline sensitivity and hypersensitization at both allodynic and hyperalgesic response ranges. This gives Ihog an unprecedented new distinct role separating it from Boi.

In total, these preliminary results hint at potential new signalling pathways apart from the canonical Hh pathway. Foremost, there is a necessity for further experimentation to truly discern the effects of Ihog and/or Boi on the induction of allodynia and hyperalgesia (i.e. is there really alteration of the severity of induced allodynia? Or is the increase we see simply proportional to the baseline increase in thermal sensitivity? And can further recordings at hyperalgesic temperatures tease out the differences that seem apparent in the distributions?). Fortunately, there is clear evidence that the exclusive absence of Ihog effects a greater increase in thermal sensitivity. This is a completely new discovery and suggests the possibility that Ihog participates in a signalling pathway distinct from the canonical Hh pathway (since its absence produces hypersensitization). The discovery of such a pathway could describe a whole new range of targets that may be viable to treat allodynia and hyperalgesia in chronic pain sufferers.

My two and a half years in Dr. Donald van Meyel’s lab, at the Centre for Research in Neuroscience at the Montreal General Hospital, has been a heterogeneous and enriching experience. I had originally been brought on as a summer student to keep stock of the fly lines, subsequently assigned to help lay down the molecular biological groundwork for the discovery/elucidation of Ihog and Boi. It is this initial experience with Ihog and Boi that led me to extend their roles into pain and sensitivity. I continue my research into pain pathology under Dr. Reza Sharif-Naeini as a Master’s in Physiology, here in McGill.

Figure 1A. Contact is made on the surface of the body wall of the larvae using the heat probe machine. The blow-up describes the canonical signalling pathway (with the addition of Ihog/boi) for Hedgehog, present in the neurons that go to innervate the body wall. 

 

 

 

Figure 1B. Response latencies between heat probe contact and rolling is a measure of the severity of the painful stimulus. The temperature used for testing allodynia corresponds to the highest temperature at which no regular larvae responds. Conversely, the temperature used for testing hyperalgesia corresponds to the lowest temperature at which all larvae respond. ns implies non-significance while asterisks (*) measure degree of significance, with *** being highly significant. We can clearly see that Ihog and Boi single mutants have a baseline increase in sensitivity (without UV irradiation). Moreover, there seems to be an increase in the severity of the induced sensitivity for Ihog mutants, which is also present after UV irradiation. In hyperalgesia, we see that there seems to be a trend for the reduction in latency following UV irradiation and a hypersensitization exclusively for Ihog single mutants. Note: UV± implies presence of irradiation. w-, boi-/- & ihog-/- describe wild-type, Boi single mutant and Ihog single mutant larvae, respectively.

Figure 1C. Theoretical mechanism for the surprising results of mutant Ihog larvae. Instead of a loss of binding due to lack of Ihog, it may be possible that Ihog constantly supresses some pathway from sensitizing neurons and that getting rid of Ihog may release the inhibition on this pathway. Note that Boi does not display similar effects.

 


References

1.              S. H. Im, M. J. Galko, Pokes, sunburn, and hot sauce: Drosophila as an emerging model for the biology of nociception. Developmental dynamics : an official publication of the American Association of Anatomists 241, 16 (Jan, 2012).

2.              D. Camp, K. Currie, A. Labbe, D. J. van Meyel, F. Charron, Ihog and Boi are essential for Hedgehog signaling in Drosophila. Neural development 5, 28 (2010).

3.              X. Zheng, R. K. Mann, N. Sever, P. A. Beachy, Genetic and biochemical definition of the Hedgehog receptor. Genes & development 24, 57 (Jan 1, 2010).

4.              D. T. Babcock et al., Hedgehog signaling regulates nociceptive sensitization. Current biology : CB 21, 1525 (Sep 27, 2011).