C. elegans Lab Report FINAL!!!!

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C. elegans Display Higher Neural
Activity When Raised in Social
Environments




Kian Bagheri
Introduction to Neuroscience
Professors Jonathan King and Elizabeth Glater
Pomona College
Tuesday November 25, 2014

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Abstract
Neural plasticity, the idea that the brain and nervous system can change in response to
external stimuli, is an important idea with implications for learning, memory, and
neurodevelopment. Caenorhabditis elegans (C. elegans) are one of the best-studied organisms in
modern neuroscience. One of the most useful aspects of C. elegans is that they are small and
transparent. Over the years, scientists have used this model organism to study many aspects of
the nervous system, most notably the development of synapses. This experiment aimed to use C.
elegans to study the effects of varying environments on their neural activity. Based on the
relevant literature, we hypothesized that C. elegans raised in an “enriched” environment—those
with more social interactions—would display more neural connectivity than those raised in an
isolated environment. This hypothesis was tested by comparing synapse development of worms
in isolated and social environments. Between one and twenty eggs were placed on growth plates
and allowed to grow for one week, after which the synapses of the developed worms were
observed under a compound fluorescent microscope. The results were consistent with the model
that C. elegans raised in an enriched environment would display more neural activity, as shown
by their more intense and frequent synapses, and higher response rate to external stimuli.

Introduction
A fundamental characteristic of the developing nervous system is that it is plastic (Rose
et al., 2005). Plasticity refers to the changes that occur in neural pathways and synapses, which
are a result of changes in behavior, neural processes, or bodily injury (Pascual-Leone et al.,
2011). Plasticity plays a fundamental role in brain repair as a result of injury, and thus its
research is important in discovering how the brain recovers from damage. It is also extremely
important in the study of neurodevelopment and learning. One of the key factors that influences
plasticity is the environment. This is especially important early in development, when the brain
grows and increases its number of synapses most rapidly (Papalia et al., 2010). Furthermore, the
effectiveness of synaptic plasticity plays a crucial role in long term potentiation (LTP), which is
thought to be one of the mechanisms underlying learning and memory (Bear et al., 2007). The
mechanism that underlies LTP is the result of a prolonged depolarization that removes an Mg
2+

block from the postsynaptic NMDA receptors. After this step, current is able to pass through the
cell, and, as a result, synaptic transmission is strengthened. How this mechanism works remains

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to be fully understood, yet by examining the development of synapses in various organisms, we
can hope to illuminate this function more clearly.
Here, we used the nematode C. elegans as a model organism to study the effects of
varying environments on synaptic development. C. elegans have a simple, well-studied nervous
system that is composed of 302 neurons (Pomona College Neuroscience 101 Laboratory Manual,
2014). Among the many advantages of studying this organism are its short life cycle, compact
and tractable genome, transparency, and small size (Pomona College Neuroscience 101
Laboratory Manual, 2014). Studying whether environmental conditions during development
affect synapses in this organism allows us to learn more about neural activity, and we can
possibly relate some of these findings to vertebrate nervous systems. We hypothesized that C.
elegans raised in an enriched environment would display more synaptic activity than those raised
in isolation. The rationale behind this hypothesis is based on the idea that deprived or abnormal
conditions cause immense disturbance in many aspects of social and emotional functioning; this
includes the growth and survival of dendrites, axons, synapses, interneurons, neurons, and glia
(Joseph, 1999). Another key factor that justifies this hypothesis is that stimulation strengthens
synapses; in isolation, the worm’s synapses are not stimulated as frequently as those raised in an
enriched environment, which results in minimal growth according to the theory of LTP.
In order to test whether certain environments influenced the development of the nervous
system, we raised worms in enriched and isolated environments for one week. The following
week we analyzed the worms’ responses to mechanosensory stimuli as well as their synaptic
growth. This enabled us to observe how different environments affected the development of
synapses in these worms. We predicted that worms raised in isolated environments would have

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less synaptic strength and fewer responses to external stimuli than worms raised in a social
environment.
Materials & Methods
After consulting the literature, an experiment was designed to test how various social
environments affect neural activity within C. elegans. The population of C. elegans that was
prepared was synchronized so that all individual worms were 7 days old (Neuroscience 101
Laboratory Manual, 2014). One large plate of CZ333 (unc25p::SNB::GFP) worms was obtained,
and eggs were isolated according to the Pomona College Neuroscience 101 Laboratory Manual.
These eggs were obtained from adult hermaphrodite worms that had eggs inside them. Bleach
was used to kill the adults, while leaving the eggs undisturbed. The worm strains had
synaptobrevin tagged with GFP. Since synaptobrevin is located on the presynaptic vesicles, the
synapses glowed under the compound fluorescent microscope.
Originally, seven plates were set up. A control with 100 eggs was created, and plates with
48, 41, 6, 4, and 1 egg were also set up in order to get a wide range of social environments.
However, when the adult worms were analyzed the following week, there were significantly
fewer worms than expected. The control had 22 adult worms, while three other plates had one,
one, and two worms, respectively. Behavioral assays and synaptic data were then collected for
all worms possible, however, the isolated worms from the two plates with a single worm died
before synaptic data could be taken. Thus, behavioral data was collected for all conditions, but
synaptic data was only collected from a single experimental condition (two surviving worms)
and the control. Behavioral assays for stimulus response and thrashing were run according to the
Pomona College Neuroscience 101 Laboratory Manual, with the only deviation being a smaller

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sample size for the isolated conditions. After behavioral assays were completed, synaptic
development of C. elegans was examined with a compound fluorescent microscope.
The images were then analyzed in ImageJ (National Institute of Health, Maryland) to
determine puncta size, density, and intensity. This was done by zooming in on the synapses in
ImageJ until they were clearly distinguishable. An oval was used to define one synapse, and the
number of synapses were counted within a 105 pixel range. ImageJ then analyzed these images
by calculating the area selected and pixel intensity. Each set of worms was analyzed in this
process, and the data obtained was compiled in a Microsoft Excel document.
Results
The objective of this experiment was to determine whether different environmental
manipulations affect the development of synapses in C. elegans. In order to observe the effects
of isolated development on synaptic strength, we ran behavioral assays and observed synaptic
traits under a fluorescent compound microscope for three isolated conditions and one non-
isolated control. Plate A had one worm, Plate B had one worm, Plate C had two worms, and the
control had more than 20 worms.
Behavioral Assays
After we observed the number of worms on each plate, we did behavioral assays for each
condition. Because there was only one worm on Plates A and B, we were only able to do n = 1
behavioral assay for those conditions. Plate C only had n = 2 worms, so behavioral assays were
done for both worms, and the control had a surplus of worms, so n = 3 behavioral assays were
done for that condition.
Although the sample size was small, worms raised in the more social control were more
responsive to external stimuli. They averaged 9.67 responses for every 10 touches to both the

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head and the tail. They also averaged 225.67 thrashes per minute when immersed in M9 buffer
(Table 1). Conversely, worms on Plates A, B, and C, who were raised in a more isolated
environment, averaged 6.25 responses per 10 head touches and 6 responses per 10 tail touches.
When placed in the M9 buffer, they averaged 101.75 thrashes per minute, less than half the
number of thrashes of the social group (Table 2).
Synaptic Data
We then moved the worms to slides and analyzed their synaptic data under a fluorescent
compound microscope. We took pictures of worms from the control group and Plate C since the
worms from Plates A and B did not survive to this stage. Synaptobrevin tagged with GFP caused
the synaptic vesicles to glow under the microscope. Therefore, brightness corresponded with
synapse size and number of vesicles. We analyzed the images collected from the fluorescent
compound microscope using ImageJ software (Figure 1).
Puncta within a span of 105 pixels were quantified and then analyzed for intensity and
size (Table 3). The worms raised in social conditions both had 12 puncta, whereas the isolated
worms had 9 puncta. The social worms also had, on average, greater area per puncta than
isolated worms. The control worms had average puncta sizes of 63-66, whereas the isolated
worms averaged 36-52. Therefore, the social worms had a greater area per puncta than isolated
worms. They also had much higher synaptic fluorescence, with intensity ranging from 697-1168
in social worms and 117-348 in isolated worms, indicating that more synaptobrevin was present
in the social worms (Figure 2). More synaptobrevin corresponds with more synaptic vesicles,
indicating that social worms have stronger synapses than those raised in an isolated environment.

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Discussion

The purpose of this experiment was to determine if isolation during development impacts
synaptic size, frequency, and strength as well as frequency of response to external stimuli in C.
elegans. Since more stimulation typically increases synaptic strength, we hypothesized that
worms raised in a more social environment would have more numerous and stronger synapses
when analyzed under a fluorescent compound microscope. We also predicted that since the
synapses of social worms would be stronger and more numerous, those worms would have
greater responses to external stimuli in a behavioral assay.
Our data supported this hypothesis. Worms raised in an isolated environment had half the
number of thrashes per minute than worms raised in a social environment, and they responded to
a tap on the head and tail less frequently than social worms. The synaptic data also supported
this, with isolated worms displaying smaller, less intense, and less numerous synapses. There
was, however, an unexpected result. There was considerable variation between individual worms
within the same condition, indicating that a lot of the variability in the synaptic data obtained
depends on the specific worm and the environment it is raised in. This could be due to
experimental error, and running more trials with a higher sample size would clarify it. Also,
synapses in our pictures, specifically with the social worms, were hard to distinguish from one
another. Synapse distinction was done by hand and therefore could have been a source of
variation in our data.
Our observation that development in isolation decreases synaptic strength supports the
finding of Rose et al. (2005) that C. elegans raised in an isolated environment had decreased
responses to external stimuli. Rose et al. (2005) also found that isolated worms were smaller and

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reproduced later in their life cycle than social worms. However, we did not observe this in our
data, as the worms in less social conditions were the same size as their social counterparts.
Our findings are consistent with reports in the literature of a stimulation-growth
mechanism for neural plasticity. Lower synaptic intensity implies less synaptobrevin, which
indicates that worms raised in isolation have fewer neurotransmitters. Rose et al. (2005) suggests
that the mechanism behind this phenomenon has to do with the glutamate receptor GLR-
1. Using GLR-1::GFP, they observed that decreased stimulation results in fewer glutamate
receptors on the postsynaptic side, and this in turn decreases the number of synaptic vesicles
needed because there are fewer receptors to stimulate. Thus, less frequently stimulated neurons
will have fewer glutamate receptors and corresponding neurotransmitters than their social
counterparts. Since our experiment only observed synaptobrevin on synaptic vesicles, it is
consistent with the GLR-1 model that more stimulation starts a chain reaction that results in
more neurotransmitters needed. An interesting direction in which to take another experiment
would be to tag the glutamate receptor and repeat the procedures followed in this lab. A
correlation between the GLR-1::GFP and SNB::GFP results would support the GLR-1
hypothesis.
Although our experiment was conducted using an organism with a fairly basic nervous
system, it has implications for many organisms of higher complexities. Scientists, psychologists,
and even parents can apply the theory of social environments enriching synaptic development to
developing youth of any species. These findings can be applied to humans, and bring up an
interesting discussion about children spending their first few days in an isolated environment.
Our experiment supports the idea that more stimulation and social interactions can help
strengthen awareness and response to the organism’s surroundings even from a young age.

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Table 1: Behavioral Data of Worms Raised in a Social Environment
Worm Condition Number of responses out
of 10 head touches
Number of responses out
of 10 tail touches
Number of thrashes
in 1 minute
Social environment:
worm 1
10 9 228
Social environment:
worm 2
10 10 225
Social environment:
worm 3
9 10 224
average 9.67 9.67 225.67


Table 2: Behavioral Data of Worms Raised in an Isolated Environment
Worm
Plate
Number of responses out of 10
head touches
Number of responses out of
10 tail touches
Number of thrashes in
1 minute
A 2 8 96
B 9 6 105
C Worm
1
7 5 125
C Worm
2
7 5 81
average 6.25 6 101.75

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Figure 1. Image of CZ333 (unc25p::SNB::GFP) raised in a social environment (left) compared to an
isolated environment (right). Images were taken using a fluorescent compound microscope at 40X
zoom. Synaptobrevin (found in vesicles near the synapse) was tagged with GFP, and thus the
synapses appear as glowing circles along the edges of the worm. Synapse size, frequency, and
intensity were measured in a span of 105 pixels using ImageJ software. In the span of 105 pixels, the
isolated worms had more numerous, brighter, and bigger synapses.


Table 3: Synaptic Characteristics of Worms Raised in a Social Environment and Worms Raised in
Isolation.

Worm Average Puncta
Size
Number of Puncta in 105
Pixels
(Average Intensity)-
(Background)
Social Worm
1
66.67 12 1168.52
Social Worm
2
63.08 12 696.95
Isolated 1 36.44 9 348.11
Isolated 2 52.89 9 117.94



105 Pixels
105 Pixels

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Figure 2. Average intensity of puncta in social worms compared to isolated worms. Intensity was
found after taking images of CZ333 (unc25p::SNB::GFP) under a fluorescent compound microscope
and analyzing them using ImageJ software. Puncta intensity was found by subtracting the
background intensity from the brightness of the synapse. This figure shows that the social worms had
brighter synapses overall than the two isolated worms. Brighter synapses correspond to more
synaptobrevin, and more synaptobrevin corresponds with greater synaptic strength.

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Literature Cited
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Glater, Elizabeth and King, Jonathan. Neuroscience 101 Laboratory Manual Fall 2014.
Pomona College, Claremont, CA.
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Papalia, Diane E. Experience Human Development. 12th ed. N.p.: McGraw Hill, 2012. Print.
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Rose, Jacqueline, Susan Sangha, Susan Rai, Kenneth Norman, and Catharine Rankin.
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