1. While previous studies look into preference behaviour to ethanol and resistant mutation to
caffeine in C. elegans, cellular and neuronal developments are currently unknown. Our
approach has been to understand the effects of ethanol and caffeine exposure at cellular and
neuronal level, especially looking at the worms developments through their life-cycle. Our
objective is to explore any regenerative effects of Vitamin D and Vitamin B on prolonged caffeine1
andethanoluse.
[1]
[2]
0
50
100
150
200
250
300
EtOH B1 CAF D EtOH+ B1 EtOH+ D CAF + B1 CAF + D Control
Counts
Samples
Adult and L4 Population Counts
High
Medium
Low
Control
0
500
1000
1500
2000
2500
3000
EtOH B1 CAF D EtOh + B1 EtOh + D CAF + B1 CAF + D Control M9
Fluorescence(arbitraryunit)
Samples
Fluorescence Intensity
High
Medium
Low
Control
M9
Effect of common household neurotoxic and neuron generating substance
on the development of nervous system in C. elegans
Abstract Methods
Results Discussion & Conclusion
References & Acknowledgments
PopulationStudy:Examining adult and L4 worms showed a decrease in population when expose to high concentration of
ethanol,vitaminB andD incomparisonwithcontrol(P<0.05,n=3).No populationchangewasobservedincaffeine,or1
its combinations (P > 0.05). However, animals exposed to both ethanol and vitamin B1 in high concentration developed a
significantincreaseinpopulationincomparisontoethanol or vitamin B alone(P<0.05).1
(Fig.1a)
Fluorescence intensity assay: Fluormeter data also agreed to a significant reduction in population of C. elegans
when expose to high and medium concentration of ethanol and vitamin B (P < 0.05, n = 3) . Animals exposed to1
bothethanolandvitaminB shownarecoveryinpopulationclosetothatofthecontrol.1
(Fig1b)
FluorescenceMicroscopy:Fluorescencemicroscopyshowed the majorityof the neurons located in the head,where
they are organised in a number of ganglia surrounding the pharynx, forming the brain. Moreover, chemosensory
neuronsmakingupalargefractionoftheheadgangliasendingtheirdendritestothetipofthenosearevisible.
Figure 1: a) Adult and L4 Population Counts. b)
Fluorescenceintensityassay.Wormsamplesbeing
exposed to Ethonol (EtOH), caffeine (CAF), Vitamin
B1 (B1), Vitamin D (D) and their combinations with
three concentrations for 1 week, were tested for
populationandfluorescenceintensity.
(b)
(a)
(f)
Figure 2: Nerves System Fluorescence
Microscopy. a,b) 20x view of C.elegans showing its
head and tail. c,d) Nervous system of C.elegans,
showing the head ganglia (brain) and the tail
ganglion. e) 40x view of head ganglia. The ganglia
forming the brain and the chemosensory neuron
withtheirextenddendritestonosie. f)40xviewof
pharnyx.
(a)
Head
Tail
(d)
Head Ganglia
Tail Ganglion
(c) Head Ganglia
· Worm samples expose to vitamin B and D suffer a significant1
reduction in population and fluorescence , which can be
related, to the effects that B and D have on the available food1
(OP50 E.Coli). Addition of vitamin B and ddH O results in the1 2
formation of non-soluble B residues that can restrict1
acceptability to food. Moreover, vitamin D itself has
antimicrobial property that allows the animal to produce an
enzymes destroying E.coli . No significant changes in caffeine
and its combinations suggest that caffeine have no significant
effectontheworms'nervoussystemandproliferation.
· In all samples, we did observe all the main head neurons.
However, no abnormal changes in the anatomy of nervous
system could be detected. This observation can be related to
strength of the controlling factors and genes affecting the
nervous system. Therefore, we can conclude that neurotoxic and
neuron generating substances used in this experiment do not
influencethedevelopmentoftheC.elegansnervesystem.
(Fig.1)
[4]
PopulationStudy:WormsampleswerepreparedwithNMG+OP50buffer
plate and 500 μl of the tested substances . These samples were
incubated at 20 C for a week. Then, the worms were analysed using stereo
microscopy at 8X zoom in three different viewpoints. One viewpoint
2
correspondedtoareaof362.6mm intheplates.
(Table 1)
mg/dl mol/L mg/dl mol/L mg/dl mol/L mg/dl mol/L
2.76 0.6 5.83 0.3 11.54 0.3 10.12 0.3
1.38 0.3 2.91 0.15 5.76 0.15 5.06 0.15
0.7 0.15 1.46 0.075 2.89 0.075 2.52 0.075
Concentrations
Alcohol Caffeine Vitamin D Vitamin B1
Fluorescenceintensityassay:DiOdyewasaddedtoeachwormplate.
After one hour, they were transferred to conical tubes for three times
washing.Triplicate100μlofeachwashedsamplesweretransferredintoa
96 well plate and their fluorescence measured using fluorometer with
484nmexcitationmax.and501nmemissionmax.[3].
Fluorescence Microscopy: Three dyed sample warms were picked
and immobilized with Agaros and microbids between microscope slides.
The fluorescence microscopy was performed with inverted microscope
at20Xand60Xzoom.
With a simple and well-characterized nervous system, C. elegans is a suitable model for studying
neurotoxicity on multicellular organisms. Here, we examined the long term effects of ethanol and
caffeine, on C.elegans and determined generative role of Vitamin D and B . Our results showed that1
1weekexposureofethanolcanimpedetheproliferationandpopulationgrowthofworms.Andthe
sameamountofexposuretoofvitaminB canreversetheimpedingeffectofethanol.1
Introduction
Special thanks to all the people
who helped us perform this
experiment:
Prof. Kilkenny
Prof. Shukalyuk
Prof. William Ryu
Chao Liu
Edward Sykes
Xi Chen
Aylia Mohammadi
Greg Stegeman
Konstantine Palanski
Jarlath Byrne Rodgers
Hang Li
[1] J. Lee, C. Jee and S. L. McIntire, "Ethanol
preference in C. elegans," Genes, Brain and
Behavior, vol. 8, no. 6 , p. 578–585, 2009.
[2] P. S. Hartman, "Caffeine-resistant mutants
of Caenorhabditis elegans," Genetical
Research, vol. 49, no. 2, pp. 105-110, 1987.
[3] "Fluorsecent dye spectra." UA Department
of Molecular & Cellular Biology. N.p., n.d.
Web. 31 Mar. 2012.
<http://www.mcb.arizona.edu/IPC/spectra_pag
e.htm>.
[4]Hewison , M.. "Antibacterial effects of
vitamin D.." Nat Rev Endocrinol. vol.7, no.6,
p.337-45,2011.
Kasra Tajdaran & Jirapat Likitlersuang
The Institute of Biomaterials and
Biomedical Engineering
University of Toronto
BME340H1
Table1.Neurotoxicandneuralgeneratingsubstanceadded to worm
samples in three different concentrations.
(e)
(b)