The effects of ethanol on Drosophila melanogaster mechanical nociception

Jennifer Le, Mariam Shehata, Isabella Slack

Department of Biology, Rutgers University, Camden, NJ. 08102

 

This study explores the relationship between pain tolerance and alcohol consumption. Alcohol consumption is linked to a decrease  in  pain  sensitivity.  Because  of  this,  sufferers  of chronic pain can self-medicate with alcohol which may lead to  alcoholism.  Conversely,  sudden  withdrawal  of  alcohol correlates with increased pain sensitivity. When alcoholics try  and  purge  their  addiction,  they  often  find  themselves more  vulnerable  to  pain  and  the  withdrawal  effects  are powerful  enough  to  draw  them  back  into  alcoholism. Previous  studies  have  shown  how  alcohol  affects the behavior of Drosophila melanogaster, a model organism, and demonstrated  that  in  adult  flies,  their  movements  become more sluggish. However, the larval stages have not been as extensively studied, especially concerning withdrawal. This study uses a Von Frey hair assay to determine whether there is a significant difference between the reaction of the larvae before and after withdrawal. The results suggest that after a period  of  steady  consumption,  the withdrawal  of  alcohol causes pain sensitivity to increase significantly and to surpass its pre-alcohol exposure level.

 

Alcohol  consumption  and  pain  sensitivity  are  intrinsically linked. Since alcohol is a type of depressant drug, it mollifies the  pain;  by  the  same  token,  the  withdrawal  of  alcohol aggravates  the  pain  (Zale  et  al.,  2015).  This  inevitably contributes to the addictive nature of alcohol since those who seek it to relieve chronic pain will experience reinforcement of alcohol consumption during withdrawal periods. Recovery from addiction is especially difficult for those in this situation because when they attempt to stop drinking, the pain returns and often seems stronger than before. It is not yet known whether the pain has surpassed its pre-alcohol levels or if it only seems that way because the individual is no longer used to the pain’s original intensity, and thus is more vulnerable to it (Zale et al., 2015). Thus, the mechanism behind how alcohol and pain affect one another is worth studying, particularly because  the  results  could  inform  how  best  to  help  those struggling with both alcoholism and chronic pain. Drosophila melanogaster is  an  excellent  model  for  investigating  the effects  of  ethanol  consumption  and  alcohol  withdrawal on nociception   because   several   research   studies   have characterized the behavioral effects of acute/chronic alcohol exposure and pain (Neely et al., 2010). To date, however, no research study has explored the impact of alcohol on pain in Drosophila.

 

While the  effects  of  alcohol  on Drosophila larvae  have  not been  studied  intensely,  the  same  cannot  be  said  about humans. Alcoholism has been associated with the failure to execute tasks or maintain healthy relationships and habits, such as work and school life (Zale et al., 2015). Despite these impaired  functions,  sufferers  of  alcoholism  continue  their routines because of the depressive effects alcohol has on pain. If an alcoholic decides to stop drinking, he or she may face the pain associated with alcohol withdrawal syndrome (Zale et al.,   2015),   which   psychologically   reinforces   alcohol dependency.  Alcohol  withdrawal  syndrome  (AWS)  can appear as soon as 6 hours after putting down a glass and symptoms   can   include   headache,   nausea,   vomiting, hallucinations,  fever,  and  high  blood  pressure  (Saitz  et  al., 1994).

 

D. melanogaster, a common type of household fruit flies, share 75% of their pain genes with humans (Milinkeviciute et al., 2012).  The  conservation  of  genes  are  incredibly  valuable when  studying  underlying  genetic  mechanisms  of  human experiences.  Further,  fruit  flies  develop  more  rapidly  than other model organisms such as mice, so more trials can be run in a relatively short amount of time. A wealth of established knowledge can be found on fruit fly behavior and genetics (Devineni and Heberlein, 2013), which means there is a solid foundation of research on which to build this study. Since much is known about the fruit fly’s life cycle, its behavior is well-characterized.

 

In  this  study,  we  used Drosophila larvae  as  the  model organism. When exposed to pain, the larvae demonstrated a 360^o lateral rolling motion (Tracey et al., 2003) which can be quantified. Third instar larvae, the most advanced stage, will be  used  because  they  are  large  enough  to  be  manipulated with relative ease, and because they are developed enough to perform the rolling motion. The rolling behavior, which we seek to quantify, does not persist into adulthood.

 

It must be noted that while the study ultimately seeks to aid sufferers of chronic pain, the rolling behavior is an indicator of nociception. Nociception is the neurological signal associated with pain, rather than the painful sensation itself. The signal travels through the organism’s spinal cord when stimulated by pain and causes a behavioral response., leading to the subjective experience of pain. The pain will be induced using Von Frey hairs.

 

Von Frey hairs (VFH) are thin filaments that are used to apply a specific amount of force. They are used in pain studies such as  the  analyses  of  allodynia  and  hyperalgesia (Jensen  and Finnerup, 2014). Respectively, the terms are a pain response to a non-painful stimulus and an enhanced pain response to an already painful stimulus. VFHs apply a specific amount of mechanical  force  with  each  application  and  elicit  a  rolling behavior from Drosophila larvae when the force is applied. The  VFH  applies  the  maximum amount  of  force  when  it buckles  under  pressure,  and  the  force  applied  is  inversely proportional to the length of the hair (Zhong et al., 2010). Therefore, the shorter the hair, the greater the force applied to  the  larva. Since  the  length  of  the  hair  is  integral  to  the response rate, the length must be adjusted to elicit a response 20%-40% of the time (Deuis et al., 2017). Although VFHs are more  known  for  their  properties  in  experiments  involving mice, the same properties can be applied to Drosophila larvae.There  is  a  possibility  that  some  of  the  larvae  will  be overstimulated  and  die  but  that  problem  can  easily  be remedied by breeding plenty of animals. 

 

Along with the threat of overstimulation, there is a possibility that ethanol can have adverse effects on the larvae. Fruit flies lay their eggs in environments with ethanol concentrations as high as 7% (Fry, 2014), so Drosophila eggs and larvae are less adversely  affected  by  the  typically  harmful  substance (Devineni  and  Heberlein,  2013) but  higher  concentrations can have adverse or even lethal effects due to the high energy cost  of  combating  the  ethanol.  One  such  effect  is  stunted growth (Castañeda and Nespolo, 2013). These obstacles must be overcome for the study to be conducted successfully and have been ameliorated in the following methods.

 

As previously mentioned, the threat of overstimulation can be remedied by breeding many animals such that the population can be split into two sets in which each set experiences a VFH treatment once. The effects of ethanol on larvae development can  be  mollified  by  inserting  the  larvae  into  the supplemented media after hatching in the non-supplemented media. This will decrease the growth stunting effect of the ethanol and will lead to the development of more third instar larvae.  Both  methods  have  been  implemented  in  the procedure.  This study seeks to determine how the exposure and  withdrawal  of  ethanol  in  fruit  fly  larvae  affect  their mechanosensory  nociceptor  sensitivity.  It  is  hypothesized that ethanol exposure decreases nociception while ethanol withdrawal increases nociception in Drosophila melanogaster larvae. 

 

Wild-type flies were graciously given by Dr. Lee’s lab. The flies were allowed to breed in a petri dish containing a grape juice agar and a yeast paste, topped with an embryo collection cage. Two embryo collection cages were made to rear two sets  of  flies:  one  that  would  be  exposed  to  ethanol,  put through a VFH treatment, and discarded, and one that would be  exposed  to  ethanol,  removed  from  ethanol  for  a withdrawal period, put through a VFH treatment, and then discarded (see Fig. 1). The collection cages were placed in an incubator set for a 12 hour day/night cycle at 22.5^oC (Pulver and Berni, 2012). A 600mL beaker full of water was placed under the collection cages to ensure that neither the agar nor the yeast paste would dry out. 

 

The petri dishes were supplied with as many flies as would fit without having to walk on one another. Then the flies were allowed two days in the chambers; one to acclimate to their new living conditions, and one to lay their eggs. After two days, the agar and eggs in the chambers were transferred to a 0% ethanol vial. Once the larvae had hatched and became mobile, they were transferred to the appropriate treatment vials  (see  Ethanol  treatment).  Approximately  5  days  were needed  between  hatching  and  the  first  VFH  treatment  to allow the larvae to reach the third instar stage (Ong et al., 2015).

 

The larvae from the collection chambers were divided equally into  eight  vials,  two  of  each  concentration.  Four  different concentrations  were  used:  0%,  5%,  10%,  and  20%.  The ethanol was mixed within the cornmeal media, on a v/v basis. The  0%  concentration  served  as  the  control  for  this experiment and was made simply by not supplementing a vial of media with ethanol. For the withdrawal portion, the second group of larvae went through a 24-hour withdrawal period, feeding on media with a 0% ethanol concentration. 

 

Petri dishes filled halfway with plain agar for moisture will be used as a platform to perform this assay. The first treatment will be administered approximately 30 minutes after the day cycle begins in the incubator. Using a VFH of a length of 0.015 m (1.5 cm), the larvae will be subjected to a poke of 1.79 g, ten times  each,  regardless  of  whether  the  poke  elicited  a response or not. The positive responses, the ones that elicited the rolling behavior, will be taken as a percentage of the total amount of applications. When this treatment is done, all the subjected larvae will be discarded.

 

After the withdrawal period, there will be a second treatment using the second group of larvae. The same VFH length will be used, and the treatment will be administered at the same time of day as the first treatment.

 

All the larvae used were from the same cohort, split into two groups:  one  that  will  be  stimulated  with  the  VFH  assay immediately after exposure, and one that will be stimulated after  withdrawal.  For  Figure  1,  a  one-way  ANOVA  was conducted  to  test  the  relationship  between  the  percent response and the applied force. For Figure 3, the student’s t-test was used to compare the results of the response of the larvae before and after withdrawal. A one-way ANOVA was used to test whether the ethanol concentrations influenced the response rates. A two-way ANOVA was conducted to see if the interaction between the two terms, the concentrations of  ethanol  and  whether  withdrawal  affected  the  response rates, were significant.

 

Figure 1. The experimental procedure asdescribed in the materials and methods. The cylinder on the far left represents the embryo chamber from which the eggs were hatched. The four rectangles represent the vials with fly food which the larvae lived and fed on. The drawings on the far right represent the petri dishes with larvae and the apparatus touching the one of the larvae is a VFH filament attached with red tape to a popsicle stick.

 

To determine the correct VFH length, tests were run where VFHs  of  uniform  width  and  various  lengths  were  used  to perform assays on larvae that were not exposed to ethanol, with the goal of finding the length at which a 50% response rate was recorded. The purpose of this was to obtain a force that  produced  a  response  rate  that  was  high  enough  to decrease with ethanol exposure and low enough to increase with ethanol withdrawal. The data reflects the larvae being successfully  poked  with  the  VFH  at  a  90^o angle,  five times. The  percent  response  was  taken  as the  amount  of positive responses divided by the total pokes. It was found that the higher force of a shorter VFH elicited more responses than the lower force longer lengths (t-test, p = 0.001788274, p < 0.05, n = 5). Since a length of 1.5 cm was used to establish a 50% response, applying a force of 1.79 g, we used the same hair for all subsequent experiments.

 

Figure 2: Von Frey hair Assay for Mechanical Nociception. The data was  obtained  by  stimulating  the  larvae  five  times,  with  the  VFH angled at 90^o. The percent responses were taken as the number of positive responses out of the total responses, regardless of whether the larvae rolled.

 

The  first  exposure  data  was  obtained  through  taking  the larvae out of their respective vials and applying the VFH 10 times and taking the fraction of the positive responses out of the total pokes. The after-withdrawal data was obtained by first  moving  the larvae  out  of  their  ethanol  supplemented vials, placing them into the withdrawal vials, and taking them out for testing after 24 hours. The testing was done in the same manner as the first exposure. Before withdrawal, the percent  responses  were  not  significant  (ANOVA,  p  = 0.209945, p > 0.05, n = 61). The 20% vial’s larvae failed to respond at all. Between only the withdrawal data, the percent responses were not significant (ANOVA, p = 0.066186, p > 0.05, n = 43). The difference in responses before and after, however, were significant (t-test, p < 0.05) for the 5% and 10 vials.  Only  the  0%  vial  and  the  20%  vial  did  not  see  a significant  difference  (t-test,  p  >  0.05).  The  interaction between  the  concentrations  and  the  before  and  after withdrawal terms were non-significant (two-way ANOVA, p > 0.05),  and  withdrawal  had  a  significant  effect  on  the  data (two-way ANOVA, p < 0.05) whereas the concentrations did not.

 

Figure 3. Percent  responses  before  and  after  withdrawal.  Within each concentration, the first exposure data are on the left and the withdrawal data are on the right. For 20%, there is only withdrawal data.  For  the  first  exposure  data,  in  ascending  order  of concentrations, the sample sizes are: n = 31, n = 17, n = 16, and n = 5. For the withdrawal data, in ascending order of concentrations, the sample sizes are: n = 20, n = 12, n = 3, and n = 8. The asterisk (*) indicate groups that have a significant difference of p < 0.05.

 

The purpose of this study was to determine what effect, if any, ethanol consumption had on nociception in D. melanogaster. The data collected from animals in the first group, those who had  been  exposed  to  ethanol  at  various concentrations without  undergoing  withdrawal,  showed  no  significant difference   in   nociceptor   activity   with   respect   to concentration (p > 0.05). Figure 3 shows that the larvae in the 5% vial rolled more than the control in the first exposure group,  contradicting  the  hypothesis  that  ethanol  inhibits nociception.This could be explained by the fact that in the wild, the larvae live and feed within an environment that is naturally rich in ethanol (Devineni and Heberlein, 2013). The same  is  not  true  of  humans,  so  the  possibility  of  ethanol inhibiting  nociception  in  humans  cannot  be  dismissed. Furthermore, the first exposure responses were lower than expected:  under  10%  across  all  concentrations.  Before conducting  the  experiment,  proper  testing  was  done  to determine an optimal VFH length, so the treatment can elicit a 40% response rate out of 10 pokes. Figure 2 depicts the results  of  this  test  and  during  the  actual  experiment,  the response  rates  were  unexpectedly  low  compared  to  what Figure 2 suggested. Genetic variation could explain the inert nature  of  the  larvae  used  in  the  experiment,  as  the  larvae were all from the same cohort. Additionally, the sample size was  low,  which  makes  it  more  likely  to  deviate  from  the average response rate. The larvae in the 20% exposure vial did not respond at all. In previous studies, larvae growing in an ethanol-supplemented environment have taken longer to develop in comparison with ones raised in an ethanol-free environment (Castañeda and Nespolo, 2013). 

 

After the initial treatment, all the larvae used were disposed. Larvae in the second set of vials were moved to control vials for 24 hours to simulate withdrawal. The percent responses after withdrawal significantly increased (p < 0.05), except for the 20% vial (p = 0.08). The larvae in the 10% vial had the sharpest increase in response but this could be due to the small sample size of n = 3. Unexpectedly, the 20% group had the least change in responses. The concentration may have been too strong and could have sedated the larvae such that they were unable to react (Scholz et al., 2000), as can occur in humans.  Lowering  the  concentration  could  increase  the response rate. The differences between withdrawal response rates were insignificant (p = 0.06), which suggests that the concentrations had no effect. For the 0% vial, before and after withdrawal,  we  did  not  expect  a  change  since  the  larvae’s conditions were not altered in any way. The data in Figure 3 reflects this (p > 0.05).

 

From the results of this experiment, one can conclude that Drosophila larvae are subject to the negative effects of alcohol withdrawal  with  response  rates  increasing  across  all concentrations. However, increasing the concentrations did not significantly chance responses before withdrawal. Due to the  low  replication  and  low sample  sizes,  a  stronger conclusion cannot be made. This experiment has also refined the VFH assay for larvae pain sensitivity, though the hair was applied manually so there could be variations in how it was applied. In future studies, the alcohol concentrations should be adjusted to ensure that the larvae are able to develop, to examine whether being exposed to the ethanol has long-term effects.

 

The authors would like to thank Dr. Lee and Dr. Fried for giving  their  unconditional  support in  this  project,  Harjit Khaira  for  helping  to  create  the  fly  media,  the  students  in group RU Afraid for supplying the grape juice agar and yeast paste,  and  Ms.  Sarah  Johnson  for  access  to  the  equipment used in this experiment. 

 

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Drosophila embryo sample preparation -OpenSPIM.