Identifying Associative Learning of C.

elegans
Jonathan Pham and Tu Vo. CMB Lab. Newman University. Wichita, KS, USA. Last Update: 4.6.2016

Abstract:
Recent studies investigating the learning paradigm in the nematode Caenorhabditis elegans have
addressed the issue of non-associative and associative learning. To test the level of associative learning
within C. elegans, this experiment will present C. elegans with a heat-shock in an environment with elevated
sodium levels. This experiment hopes to observe the C. elegans ability to associate the heat-shock with the
abnormal sodium levels in their environment. There must be physiological changes taking place at the
synaptic or sensory neuron level in order for association between the processes of chemotaxis and
thermotaxis to occur. Further research beyond this experiment into the complex biochemical pathways
involved in associative learning of C. elegans may provide insight into the pathways that humans use.
Introduction:
The ability to learn can be defined as an
adaptive change in behavior in response to
environmental stimuli. 12 Hence, this experiment
assumes that observed change in behavior
indicates learning. Despite having approximately
only 210 neurons in their head, the C. elegans
worms have displayed some ability to learn.13 The
structure of the nervous system of C. elegans
consists of about 300 neurons overall, 5000
chemical synapses, 600 gap junctions, and 2000
neuromuscular junctions.13 Of the C. elegans
neurons 300 neurons, there are 118 classes of
neurons: 48 interneurons, 30 motor neurons, and
40 sensory neurons.14 The sensory neurons allow
thermotaxis as well as chemotaxis, giving worms
the ability to sense heat and nearby food and
toxins. 14
One of the first studies to research the
learning and memory characteristics in C.
elegans was done in 1990. This study analyzed
non-associative learning of the organism by
observing reflex responses to a mechanical
stimulus. According to the study, C. elegans is
capable of short-term habituation, dishabituation
and sensitization. 10A more recent study done in
2010 reported, It [C. elegans] has since proven
to be exquisitely sensitive to its environment,
displaying remarkable behavioral plasticity. 2
Based on these studies, C. elegans should possess
a level of short-term memory capable of
associative learning. Associative learning can be
described as an animals ability to link one
stimulus or behavior with a second stimulus or
behavior. 12
Synaptogenesis is a fundamental process
that must occur for any type of associative
learning to take place. In the scientific world
today, the biochemical pathways that underlie
synaptogenesis are complex and incompletely
understood.9 Through this process, an
organisms neurons are able to form specific

connections to their targets through synaptic

formation. 4 According to neurobiologist Yishi
Jin, synaptogenesis involves the formation of the
neurotransmitter release site in the presynaptic
neuron and a receptive field at the postsynaptic
partners, and the precise alignment of pre- and
post-synaptic specializations. 6 Describing
vertebrate nervous systems, Jin writes, synaptic
activity plays a crucial role in shaping synaptic
patterns. Not much is explored to address
whether and how much activity plays a role in C.
elegans. 6
If C. elegans can exhibit associative
learning by correlating heat with higher
concentrations of NaCl, then it can be understood
that their neural complexes will have changed in
some way. If the behavior of C. elegans is
expressed by its sensory neurons directing to its
motor neurons mediated by interneurons as Xin
and Deng suggest,14 then the observed behavioral
change is likely due to 1) the formation of new
synapses or 2) the interneurons that receive inputs
from chemotaxis release signals to effector motor
neurons as if it received inputs from chemotaxis
and/or thermotaxis. Otherwise, the observed
behavioral change is likely due to a
complimentary release of neurotransmitters
and/or action potential whereby the excitation of
a chemotaxis neuron leads to the excitation of a
thermotaxis neuron. To observe these pathways,
we hope that attaining evidence of C. elegans
associated learning of heat and salt will open a
new window of opportunity for research
regarding the development of neuronal
complexes.
In C. elegans, ASE gustatory neurons
are capable of sensing salts and other watersoluble attractants. The ASER (the right neuron)
senses potassium and chloride ions, while the
ASEL (the left neuron) can detect sodium ions.3
Heat avoidance, on the other hand, is related to
nociception: the ability to process harmful

stimuli. Studies indicate that the transient

receptor potential (TRP) channels, as well as the
FLP-21/NPR-1 neuropeptide signaling pathways
are involved in heat avoidance for C. elegans.5
According to a study concerning the model of
chemotaxis and associative learning in C.
elegans, the ASEL and ASER neurons are not
coupled by gap junctions nor chemical synapses,
due to their functional asymmetry.1 The study
stated, One possibility is that learning takes
place at the synapses between the ASE neurons
and the interneuron.1 Various studies have been
done to test C. elegans associative and
nonassociative learning ability, but there have yet
been a study attempting to induce direct
associative learning of chemotaxis and
thermotaxis. However, should C. elegans show
evidence of associative learning between their
chemotaxis and thermotaxis, then the
development of associative learning can be
further studied with greater insight and
specificity.
A study by Li et al showed that
serotonin may regulate memory of thermotaxis
neurons via G-protein coupled serotonin
receptors.6 Here, memory is defined as the coding
and storage of pre-existing messages and is
encoded by a number of neurons such as AFD,
the thermal sensor, and AIY, the complementary
interneuron.6 Mutations of serotonin production
or regulation genes have shown to result in
deficits of thermotaxis memory in C. elegans.7
The paper by Ikue Mori suggests that the
intracellular messenger for the sensory systems of
C. elegans, cGMP, uses a G-protein signaling
cascade to relay messages and the process
demonstrated similarity with vertebrates.8 Thus,
understanding the pathway(s) in which C.
elegans develop its learning ability can reveal the
pathway(s) in which humans and like-animals
develop their learning ability.
In light of C. elegans ability to respond
to its environment, as well as its short-term
habituation, we believe that we can observe
associative learning occurring within the species.
The two stimuli we wish C. elegans to associate
with each other are the elevated sodium levels
and the stressful heat-shock that will be applied
to them. We hope to observe the C. elegans
association with heat stress and sodium by
avoiding environments with abnormal sodium
levels.
Hypothesis:
If the C. elegans undergo heat-shock in a
culture prepared with sodium, then they will be

unlikely to travel to a food source with sodium as

compared to a food source without sodium.
Materials and Methods:

N2 C. elegans

6 regular plates seeded with E. coli

6 plates seeded with E. coli and contains

+NaCl (+ .5mL of [#] M NaCl)
10 hybrid plates with 2 E. coli lawns on
opposite side; 1 with +NaCl (+.1mL of [0.683] M
NaCl)
Before beginning the experiment, E. coli
and C. elegans were tested at various
concentrations of NaCl and parameters of heatshock for optimal observations. The parameters
of heat-shock for optimal observations were 3
minutes under varying temperatures: between 50
to 80 degrees celsius. After testing, 65 *C under 3
minutes were determined to be the appropriate
parameters. The addition of NaCl onto the agar
plates were 0.1mL of [0.683] M +NaCl to one
side of the hybrid plates and 0.5mL of [0.683] M
+NaCl to the salted plates.
A culture of C. elegans were age
synchronized as instructed by the Maintenance
of C. elegans written by Theresa Stiernagle.11
The L1 worms harvested from age
synchronization were then transferred equally to
an E. coli OP50 lawn. This procedure was used to
prepare all synchronous cultures of C. elegans
necessary for this experiment and the quantity
varied based on resources at the time and based
on desired number of samples.
Once the worms have reached the L4
stage, worms from one of the age synched plates
were washed by 0.5mL sterile H2O and
transferred to a plate seeded with E. coli lawn and
contained +NaCl. The plate then underwent heatshock, exposing to the worms to a high-stress
heat event. The heat-shock consisted of
undergoing 65 *C exposure for 3 minutes and the
heat was applied from underneath the agar with a
hot-plate. The C. elegans that underwent heatshock were then immediately transferred to a
hybrid plate containing two pools of E. coli on
opposite side by chunking: one end containing
+NaCl and one without. The chunked worms
were placed at the center so it may travel to either
E. coli lawn at opposite ends of the agar.
Observations were noted 10-15 hours later by
counting the number of worms in each lawn. This
was repeated many times more to get the desired
sample size.

Finally, a chunk of worms from the age

synched plate was placed in the middle of a
hybrid plate. This would allow a control for
analysis of the C. elegans innate tendency to

prefer area with +NaCl and area without +NaCl.

This was repeated once more from the same age
synched plate.

Fig. 1: The entire process is summarized on this diagram.

Results:
If our hypothesis is correct, the C. elegans would avoid the E. coli lawn with +NaCl. Because
studies have shown that C. elegans to have some form of associative learning, we expect our hypothesis to
be true. Avoidance is observed based on the number of C. elegans counted in each lawn of E. coli compared
to the other. Analysis is done by calculating the ratio of C. elegans in each E. coli lawn and then interpreted
based on two chi-square tests. Insofar, the data supports the hypothesis by rejecting the null hypothesis in
both instances. Rejection of the null hypothesis indicates that the data of the experimental plates show no
significant similarity with the control plates nor with the base assumption that the C. elegans would travel to
either side of the hybrid plates equally. Hence, data has so far indicates that there is a change in behavior as a
result of the heat shock; thus, associative learning of thermotaxis and chemotaxis of C. elegans is concluded
viable based on this first experimental trial. However, we must replicate this experimental design several
more times before we can reach a conclusive statement.

5
6

3
3

NaCl+/regular
ratio
1.666666667
2

Total:

11

1.833333333

Plate 1
Plate 2
Plate 3
Plate 4
Plate 5

1
3
12
10
4

0
12
33
15
11

Plate
Control 1
Control 2

# of worms in +
NaCl

# of worms in regular
agar

When Null =
1.83333
X^2

When Null = 1
X^2

1.367424
1.178187
0.742424
1.178187

0.5625
0.404959
0.111111
0.404959

NA
0.25
0.363636364
0.666666667
0.363636364

P-

P-

Total:

29

71

0.408450704

4.466222

value
0.198

1.483528

value
0.687

Fig. 2: The null hypothesis of the first chi-square test assumes that 50% of worms would travel to +NaCl relative to no +NaCl. The
null hypothesis of the second chi-square test uses the % of worms that traveled to the +NaCl relative to the side without +NaCl while
having the null as the number observed on the standard examined in last procedural step. The second chi-square test eliminates the
bias of C. elegans that may instinctively prefer higher or lower NaCl concentration and is the primary test for interpretation.

Significance of work:
This experiment has the potential to provide further insight as to whether C. elegans has the
capacity to learn to associate two different biological pathways. If we investigate the extent of these neurons
capacity, it would serve to promote further studies regarding neuroscience. The learning ability of C. elegans
could then be compared to that of higher species.
A further and more extensive study into the biochemical pathways involved in associative learning
and synaptogenesis in C. elegans can give more insight on how the same pathways work in humans. Gene
function of neurons within most species is for the most part evolutionarily conserved, which is why the study
of C. elegans is significant for research on human development and function, including neuroplasticity,
motor and behavioral development. In addition, if the experiment is successful, scientists can set up
experiments to study which of the three pathways aforementioned are taking place in the process of adapting
associative learning and synaptogenesis.
Limits of experimental design:
C. elegans may be instinctively inclined to move to an area with +NaCl or to an area without
+NaCl. Controls were made to handle this bias. On the other hand, the +NaCl concentration were selected
based on E. coli and C. elegans ability to survive in the concentration, not by its reaction towards the
concentration. Thus, the +NaCl may not be sufficient to suggest significant results. Nevertheless, if there are
significant differences between the independent plates and the control plates, then the +NaCl concentration
is assumed sufficient for the worms to sense.
Various errors were observed when carrying out experimental steps, including that most of the C.
elegans would not travel to either food source and remained near or in the chunk, creating low sample size
and possibly erroneous ones. This experiment has only been carried out once and we look to repeat the
experiment several more times before we can reach a conclusive statement.
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