Brain and Behavior Student Manual 2022

VISION

LABORATORY 7
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SUMMARY
The goal of these experiments is to allow you to examine how the visual system generates a rich
perceptual representation of the world, and how perceptual phenomena relate back to the
neural organization of the visual system. You will complete four exercises exploring (1) visual
acuity, (2) color vision, (3) visual illusions, and (4) visual plasticity.
OBJECTIVES

• Understand how to measure visual acuity and how it relates to the organization of the
retina

• Be able to explain current theories of color vision and “color blindness”
• Understand why visual illusions occur, and what each tells us about the workings of the

visual system
• Explore how transient plasticity arises in the visual system

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INTRODUCTION

The primate visual system is one of the most complex and intriguing sensory systems, and the
one we understand in the most detail. Perception of shape, movement, color, depth, and fine
spatial detail are just some of the many visual phenomena we experience. In the vertebrate
retina, photoreceptors (rods and cones; see Figure 1) use light energy to transmit sensory
information in the form of neural signals to the ganglion cells, which carry the information to the
visual nuclei of the thalamus, from which it is transmitted to the visual cortex and beyond to
other parts of the brain.

Figure 1. The photoreceptors of the retina (rods and cones), located at the back of the eye.

Retinal Composition

There is a minimum amount of stimulus energy required for the perception of a stimulus, referred
to as threshold. As we saw during the somatic sensation lab, we can use a measurement of acuity
to quantify threshold. Visual acuity is the finest spatial separation of two visual stimuli that can
be resolved as two objects rather than one, or, as commonly measured in the eye clinic, the
smallest representation of a letter that can be correctly identified.
Acuity is determined by the two classes of photoreceptors found in the primate retina: rods and
cones. Rods are responsible for night vision and motion perception, while cones are responsible
for the perecption of fine details and color. The center of the retina, an area known as the fovea,
is densely packed with cones, while the periphery contains mostly rods. As a result of this
organization, when we fixate directly on an object, the light stimulus falls onto the centrally
located cones, while information in the periphery is translated by the less sensitive rods.

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Figure 2 [HANDOUT].
Organization of the retina.
Note how the central fovea is densely
packed with cones, while the periphery
has a greater proportion of rods.

Color Vision

In addition to enabling vision for fine detail, cones are also critical for the perception of color.
Humans have three classes of cones, differing in the particular photopigments they contain.
Currently there are two main theories of color vision. According to the Trichomatic Theory, each
photoreceptor responds maximally to a different range of wavelengths of light: long wavelengths
(reds), medium wavelengths (greens), or short wavelengths (blues). Colors can be represented
by a balance of activation in the three cone types.

Building off this theory, the Opponent-Process Theory suggests that the three cone types are
linked together in pairs: red-green, blue-yellow, and black-white. When one is activated, it
inhibits the other color (the ‘opponent’) in its pair. For example, we would see yellow by the
mixed activation of the medium (green) and long (red) cones, which inhibit the short (blue) cone,
resulting in the perception of yellow. However, if light in the blue wavelength range is present,
the short cone is activated and it will inhibit the other cones. This is sometimes refered to as
“winner take all” since the cone that is most activated will be able to exert the greatest inhibition
of its paired cone, as if the colors are competing to be represented.

The interactions between the cones is especially apparent in experiments with adaptation, as
you’ll see today. Recall that neurons can change their firing rate to reflect the state of the
environment. One can imagine that constant activation of one of the cone types would lead to
adaptation, such that it would not need to continuously fire at a high rate to signal the strong
stimulation. However, if a weaker stimulus is then provided, the cone that experienced
adaptation will not respond as well. So, in relation to the “winner take all” competition among
cones, the adapted cone would exert less inhibition on the opponent colors, which allow the
opponent color cones to then rebound from their continuous suppression. The resulting
perception will then be one which emphasizes the ‘rebounding’ colors.

The process of mutual inhibition of connected photoreceptors is known as lateral inhibition, and
is crucial not only to color vision, but also in the emphasis of contrast and edges. Today in lab,
you will be exploring color opponency and lateral inhibition using a computer demonstrations.

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Colorblindness

Variation in the distribution of photopigments among individuals is normal, but some people
may have quite different pigments or may be lacking one or more of the cone types. Today you
will use the Ishihara Color plates to test whether or not you are “color normal.” In these plates,
a figure or pattern made up of a series of dots of one or more colors is embedded in a
background of differently colored dots. The only systematic difference between the figure and
the background is color. Observers with normal color vision should have no trouble picking out
the embedded figures; individuals with color deficits may not be able to see all of the figures.
While not a definitive determination of color vision, this test is used as a screening tool to help
identify subjects who are “colorblind.” It is important to recognize that the term colorblind here
refers to many types of color abnormalities, not just the extreme condition where there is no
sense of color at all (known as achromatopsia).

The most common congenital anomalies are red-green; about 10% of males show these inherited
red-green anomalies which have X-chromosome linked recessive inheritance. Other individuals
may show blue-yellow color difficiences. The paired nature of color blind vision was one of the
key pieces of evidence for the Opponent-Process Theory.

Visual Illusions

The visual system is highly efficient, quickly generating rich perceptions from patterns of light. As
scientists, we can utilize visual illusions to observe the complex nature of the interaction
between the environment and the visual system. In today’s lab, you will have a chance to explore
several types of visual illusions, all of which try to trick the system in order to expose the
principles by which vision normally operates. Illusions demonstrate that the visual system does
not only translate the direct stimulus inputs to form a percept, but rather depends on
interpretations and basic rules of how the world tends to be organized. Below you will find a
description of some of these principles.

• Visual illusions are often taken as evidence that “the whole is greater than the sum of
the parts,” indicating that the visual system tends to take the overall summary of the
information provided from a stimulus, rather than attending and utilizing every detail.
The resulting perception comes from the visual system trying to put together similar
information and interpret it as a unified whole.

• Our perceptions are not only based on the information from the stimulus, but
interactions with previous experiences, such as previous activations of the same parts of
the system. In addition higher level congitive experience impose other interpretations,
such as interpreting perspective based on the scenery one is used to experiencing.

• Overlapping objects in the environment indicate depth, with the occlusion of one object
with another indicating that one object is behind the other.

In the following exercises you will see how these assumptions can also ‘trick’ the visual system.

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Plasticity

In general, plasticity refers to the way that the brain can change in response to experience.
As you’ve been learning all semester, the connections between neurons allow for information
processing and the generation of behaviors, and it is these connections that plasticity often
targets. Plasticity is especially common during brain development, with early experiences
permanently shaping the formation of neuronal connections. However, the brain does not only
undergo plasticity during development. After brain damage, plasticity confers a resilency on the
brain, such that the non-damaged areas form new connections to compensate for injury and tend
to take over functions previously carried out by the damaged regions.

Plasticity is not only seen in the extreme cases of development and damage, but can also involve
smaller scale changes, for example, in response to learning new information. In this way, we are
able to form memories and draw on the previously learned information at a later time point.
In addition, plasticity can also be seen on a transient (short-term, often reversible) time scale.
Today, you will explore an example of plastic changes that can rapidly occur in response to a shift
in visual perception by wearing goggles that shift the visual input from the world. After limited
experience, you should be able to see the visual system adapt, and once again be able to
efficiently interact with the world. You will test the resiliency of plasticity by re-examining your
performance once the goggles are removed.

Eye Chart

Doctors use eye charts to measure how well someone can see in the distance during checkups
and eye exams. The doctor will ask you to stand about 20 feet away from the chart and read the
smallest line of letters that you can make out. From there they will be able to accurately define
the farsightedness of your vision. A classic example of an eye chart is the Snellen eye chart,
developed by Dutch eye doctor Hermann Snellen in the 1860s (Figure 3).

There are many variations of the Snellen eye chart, but in general they show 11 rows of capital
letters. The top row contains one letter, usually a letter “E” but other letters can be used.

The other rows contain letters that become progressively smaller and smaller as you move down
the chart.

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Figure 3. Snellen Eye Chart. Image retrieved from http://www.cascadilla.com/eyecharts/

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Brain & Behavior: CORE-UA 306

LABORATORY COVER SHEET
Name: _____________________________ Lab Partner’s Name: _________________________
Date of Lab: ___________________________ Date Due: _______________________________
Lab Instructor: _____________________________ Lab Section Number: __________________

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Procedures and Exercises

You will be working in pairs for this experiment. Both partners will have a turn as the
experimenter and subject.

PART I: VISUAL ACUITY – The Blind Spot

The blind spot is a part of the retina where no photoreceptors are present. To demonstrate its
existence to yourself, in the following exercises you will be able to detect your own blind spots
and even measure them. While working through these exercises, you should consider how acuity
relates to the organization of the retina.

PROCEDURES:
A. Materials needed to test your blind spot:

• 2” x 5” Cardstock paper
• Pen, pencil, or marker
• Ruler
B. Preparing the Blind Spot Test:
1. Place the paper rectangle on a surface so that it is oriented lengthwise i.e., the 5” edge

nearest you.
2. On the left edge of the paper, halfway between the top and bottom, draw a small shape

(no wider than half an inch) such as a circle, star, or plus sign. Use the ruler to gauge ½”.
3. On the right edge of the paper, halfway between the top and bottom, draw a different

shape of approximately the same size.

C. Testing your Blind Spot:
While one student (the SUBJECT) performs the steps of the blind spot test, their partner (the
EXPERIMENTER) will need to read the steps aloud and jot down the subject’s immediate
responses to the italicized questions below the steps. This DATA will be useful to answering
QUESTION 1.

STEP 1 - Hold the middle of the rectangle in your right hand; make sure both shapes are visible.
STEP 2 - Fully extend your right arm with the paper at eye level. Focus both eyes on the left shape.
DATA ENTRY 1: With your eyes still focused on the left shape, can you still see the shape on the
right side of the paper?

STEP 3 - Slowly move your extended arm closer to your face. While moving the paper closer, keep
both eyes focused on the left shape.
DATA ENTRY 2: While moving the paper, can you still see both shapes clearly?

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STEP 4 - Cover your left eye with your left hand.
STEP 5 - Fully extend the right arm with the paper at eye level again. Focus your right eye on the
left shape.
DATA ENTRY 3: Can you see the other shape as well?
STEP 6 - With your left eye covered and your right eye focusing on the left shape, slowly move
the paper closer toward your face. Keep focusing your right eye on the left shape.
DATA ENTRY 4: What happens to the shape on the right side of the paper while you move the
paper closer?

STEP 7 -Now cover your right eye with your right hand.
STEP 8 - Extend your left arm with the paper and look at the right shape.
DATA ENTRY 5: Are you able to still see the left shape while focusing on the right shape with
your left eye?
STEP 9 - Again, slowly move the paper closer to you. Keep your left eye focused on the right
shape.
DATA ENTRY 6: What do you notice this time?

QUESTION 1:
A. Describe the responses you recorded while performing the blind spot.
B. At one or more of the steps in the procedure you should have observed that one of the shapes
“disappeared” from your view. How can you explain this phenomenon based on what you know
about the organization of the retina?

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PART II: COLOR VISION

Next, we will use a screening test called the Ishihara Color Plates to examine color vision.
On each card there will be a figure or pattern made up of a series of dots of one or more colors,
embedded in a background of differently colored dots. The figure and the background differ only
in color, a difference that will only be difficult to perceive for those with abnormal color vision.

PROCEDURES

Both partners should complete this task at the same time, quietly making note of their
observations.

1. For each plate number, record if you see an embedded figure or pattern in the space below.
If you cannot see any pattern, be sure to record that as well.

2. Compare your responses to the “standard” set. If your responses match, you are color
normal. If they do not match, check in with your lab instructor to see if there is a particular
pattern to the differences.

Book Number: Color Visual Screening Actual Figure
Plate Number
Response

QUESTION 2:
How can the composition of the retina account for abnormalities in color vision?

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PART III: SIZE, SHAPE AND COLOR ILLUSIONS

You will be exploring a few visual illusions in this section, learning how the context a visual
stimulus is embedded in can alter how it is perceived. In the first example, the Müller-Lyer
Illusion, you will experience an illusion of size perception. You will also look at several color
illusions, which should help you to understand the underlying processes leading to color vision.
The After Images illusion will demonstrate what happens to the mutual inhibition when there is
continual activation of certain set of cones. Finally, the Color Context illusion will demonstrate
how color perception is based on a balance of foreground and background information.

 The Müller-Lyer Illusion

Work in teams of two during this exercise, with one student acting as the “subject” and the other
student acting as the “experimenter.” Once the first subject performs 6 trials, the subject and
experimenter will switch roles and perform another 6 trials.

Figure 4. Screenshot of Müller-Lyer Illusion webpage.

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PROCEDURES

The “Müller-Lyer Illusion” webpage has been bookmarked on all laptops in your lab room (see
Figure 4). If you are unable to locate it, type in the following url:
http://www.michaelbach.de/ot/sze_muelue/index.html

1. The subject should drag the middle arrow ( < ) until it is in the middle of the line.

2. The subject will then close their eyes while the experimenter clicks “show result.”
The experimenter should record the percent off-center measurement in the chart below.
Then the experimenter will click “reset.” Do NOT tell the subject his/her results.

3. Repeat the procedure 6 times; record results of each trial on the chart labeled Naïve Subject
below.

4. Switch roles. For this set of trials, the former experimenter will act as a non-naïve subject
since they are already familiar with the experiment. Repeat steps 1-3 (even as the non-naïve
subject, it is still crucial that the subject not know their results after each trial). Record results
in the Non- Naïve Subject chart below.

Naïve Subject Non- Naïve Subject

Trial # % off-center Trial # % off-center
measurement measurement

11

22

33

44

55

66

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Read through the ‘perspective explanation’ on the website. This explanation suggests that the
illusion arises because shapes with angles “in” (<-->) are typically found in the front of a stimulus,
and are interpreted as closer, while those with angles “out” (>--<) are normally found on the far
end of a stimulus, and further away. Closer objects are often larger when they fall on the retina
than objects that are farther away. However, in the illusion, the two lines have exactly the same
retinal size.
QUESTION 3:
A. How do your results compare with this explanation?
B. Does this phenomenon occur similairly in the naïve and non-naïve conditions?

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 Color

All our sensory systems utilize past experience and surrounding context to interpret new sensory
input. In the next two exercises, you will explore how these apply to the visual system, and
specifically to color vision. Recall the Opponent-Process Theory: the three cones, each sensitive
to a different wavelength of light, interact through inhibitory connections to generate the
perception of color.

PROCEDURES

1. Use the “Color Adaptation 1.1” and “Color Adaptation 1.2” Stare at the fixation cross in the
middle of the four eagles for at least 1 minute. This should be sufficient viewing time, but
longer viewing will strengthen the effect.

2. Proceed to the next slide, and try to focus on the afterimage of one eagle at a time.

QUESTION 4:
Jot down your first responses on the charts below.
A. What color did you see for each eagle on the first slide?
B. On the second slide?

Color Adaptation 1.1 Color Adaptation 1.2

QUESTION 5:
What neural property underlies the after image phenomenon?

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QUESTION 6:
A. Why is it necessary to stare at the fixation cross in the first part of the experiment?
B. What do you think would happen if you move your eyes around instead?

Refer to the “Color Interaction” slide shown by your lab instructor.

1. Look closely at the colors of each word. List the color you see for each word in the chart
below:

Color Interaction Observations

Word Color
Annual

Review
Of

Psychology

2. Now look carefully at the small connecting bars between Annual and Review and between Of
and Psychology, then answer the following questions.

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QUESTION 7:
A. What do these connecting bars demonstrate about the colors of the words Annual and Review
and between Of and Psychology?
B. How does this compare to the observations you listed above?

QUESTION 8:
What does this experiment tell you about how the visual system interprets stimuli?

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PART IV: VISUAL PLASTICITY

In this experiment you explore transient visual plasticity, which in this case will be triggered by
goggles that will shift the visual world by about 30 degrees. It’s important to remember that while
we discuss each sensory system separately, there is a constant coordination between all your
senses. As you gain experience with the goggles, both the visual and motor systems will adapt to
efficiently accomplish the task at hand.

Activity Description

This exercise must be done with a partner. For baseline measurements, without the goggles on,
the subject will toss 4 bean bags at the target (flat on the floor) for each trial from approximately
4 feet (~ 1.2 m). The floor grid is a series of parallel lines, the center of which is the target (Figure
5). The accuracy of the tosses will be measured by the experimenter. The subject will then put
on a pair of goggles, which will alter his or her visual perception. After a series of trials with the
goggles on, the goggles are removed and performance is measured again.

Target line

-4 -3 -2 -1 +1 +2 +3 +4

Figure 5. Visual Plasticity Grid

QUESTION 9:
Hypothesize how your performance will change, if at all, (A) immediately after you put the
goggles on, (B) after several trials with the goggles on, and (C) immediately after taking the
goggles off, when you return to normal vision.

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Visual Plasticity: Procedures

BASELINE RECORDING:
1. The first subject should stand at/behind the “toss line” - about 4 ft. from the target.
2. Toss the 4 bean bags (one at a time) towards the center of the target.
3. The experimenter will use the table on the next page to record the value for landing
location of each bean bag under table column heading “Normal.”

NOTE: Record where the bean bag lands, not where it ends up after sliding. The values for the
grid can be seen in Figure 5. If the bean bag lands to the left of the center line, the values recorded
are negative; to the right, they are positive.
GOGGLES ON TRIALS:

4. Subject 1 will put on the Plasticity goggles and repeat tossing the 4 bean bags, one at a
time. Do not remove the goggles between each trial.

5. The experimenter will record the landing location of each bean bag under the table
column headings for “Goggles on” and retrieve the bean bags after each trial.

6. Subject 1 must repeat the 4 tosses for each of the 3 trials – 12 tosses in total.
GOGGLES OFF TRIALS:

7. Once the first 3 trials are completed, Subject 1 must remove the goggles and immediately
repeat the exercise for 3 more trials (which is another 12 tosses).

8. The experimenter will record the landing location of each bean bag under the table
column heading for “Goggles off.”

If there is sufficient time, switch roles and repeat the exercise so that each partner contributes a
set of data. Enter results in the tables on the next page, then complete the Average Values table.

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Bean Bag Toss Accuracy

Condition Normal Goggles on, Goggles on, Goggles on, Goggles off, Goggles off, Goggles off,
trial 1
Subject 1 trial 2 trial 3 trial 1 trial 2 trial 3
TOSS 1
SUBJECT 1

Subject 1
TOSS 2

Subject 1
TOSS 3

Subject 1
TOSS 4

Condition Normal Goggles on, Goggles on, Goggles on, Goggles off, Goggles off, Goggles off,
trial 1
Subject 1 trial 2 trial 3 trial 1 trial 2 trial 3
TOSS 1
SUBJECT 2

Subject 1
TOSS 2

Subject 1
TOSS 3

Subject 1
TOSS 4

Take an average of all values associated with each condition of bean bag toss for both subjects.

AVERAGE VALUES

Condition Normal Goggles on, Goggles on, Goggles on, Goggles off, Goggles off, Goggles off,
trial 1 trial 2 trial 3 trial 1 trial 2 trial 3
Subject 1

Subject 2

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Plot the average value obtained for each trial, for both subjects, on the graph below.

GRAPHICAL PLOT OF VISUAL PLASTICITY

QUESTION 10:
Re-evaluate your hypotheses. How did your performance after a few trials with the goggles on
compare to your performance when you took the goggles off?

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QUESTION 11:
A. How many trials did it take for you to see evidence of plasticity when the goggles were on?
B. What about when you took the goggles off?
A. GOGGLES ON:

B. GOGGLES OFF:

In the previous experiment you saw that the visual system is able to rapidly adapt to new
conditions as extreme as a 30 degree shift in perception. With each trial you should have been
able to see an improvement in performance, as you were able to gain experience with the
goggles, and then again without them. However, on each trial you also received feedback from
the visual and motor systems about your performance.
QUESTION 12:
Hypothesize how feedback could interact with plasticity (i.e. what do you think would happen if
you could not see the outcome of your beanbag toss).

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CONCLUSION QUESTIONS

After completion of the lab, you should be able to answer these conclusion questions.
QUESTION 13:
When the Müller-Lyer Illusion was tested in rural villages, it was not sucessful. However, it almost
always works in Western societies. It is hypothesized this might be because the architecture in
these villages are rarely made of 90 degree angles, which form the basis of the illusion.
A. What does this suggest about the relationship between experience and the visual system?
B. How might this come about in the brain?

QUESTION 14:
Throughout today’s lab you’ve seen how the visual system creates the illusion of perceptions
beyond what is actually present in the stimulus itself. Name two examples from today’s lab and
explain how this could be considered helpful for the survival of an organism in a changing
environment.

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QUESTION 15:
Babies are not able to fully interpret perspective or depth information from the environment
until they are a few months old; for example, they will not be able to understand an occluded
object does not simply have gaps in it. What do you think underlies the development of visual
perception?

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CLEAN-UP PROCEDURES

When you are done with your work, you must clean up. You will not be permitted to leave the
lab until the lab instructor checks your lab bench.
Please leave your lab station as clean and orderly as when you arrived.
Before leaving the lab, double-check your area for your personal belongings.
THE LAB STAFF IS NOT RESPONSIBLE FOR ANY LOST OR STOLEN ITEMS.
Failure to properly clean up will result in a 5-point deduction from your laboratory assignment.

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OBSERVING BEHAVIOR: Learning and Memory with The Model Organism Caenorhabditis Elegans

LABORATORY 8
OBSERVING BEHAVIOR: Learning and Memory with

The Model Organism Caenorhabditis Elegans

SUMMARY
The purpose of today’s laboratory exercise is to illustrate how to use scientific experiments to
quantify and learn from observable behaviors. In particular, you will be examining how organisms
interact with, and learn from, their environments in order to shape future behaviors and promote
survival. Today you will observe the innate behaviors of Caenorhabditis elegans (abbreviated as
C. elegans) and examine how complex behaviors such as learning and memory can be studied in
an anatomically simple model organism.
OBJECTIVES

 Understand the methodologies used in behavioral neuroscience
 Become familiar with principles of experimental design for behavioral analyses
 Observe and learn to recognize C. elegans’ innate behaviors and responses to

environmental influences
 Quantify learning and memory in C. elegans

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INTRODUCTION

The ultimate goal for all species is to optimize survival. Across generations, evolutionary
adaptations allow organisms to better respond to (and thrive in) their environment, while within
a single lifetime, individuals will learn new behaviors to enhance their chances of survival.
To do so, organisms must be able to extrapolate information from their experiences and apply
that information to future experiences. For example, after being stung by a bee, a child may
become wary of insects; the learned relationship between a bee’s sting and pain allows the child
to avoid being stung in the future. The neural basis for the formation of learned relationships is
the primary focus of Behavioral Neuroscience.

C. elegans as a model organism

In this lab, you will act as Behavioral Neuroscientists, exploring the innate and learned behaviors
of Caenorhabditis elegans (C. elegans), a small worm that displays many of the same acquired
behaviors as larger animals with more anatomically complex brains, such as mammals.

C. elegans are soil dwelling, bacteria-eating nematodes (roundworms) that are approximately
one millimeter in length. They have a relatively short lifespan and reproduce quickly, making
them ideal experimental subjects. The C. elegans nervous system is composed of exactly 302
neurons, all of which have been identified, and scientists have been able to fully map their neural
circuits (the connections between different neurons). As such, the adult worm’s nervous system
is an attractive model system for linking neuronal and molecular function with behavior.

While the anatomy of C. elegans is quite simple, the cellular components and molecular
mechanisms of its nervous system are similar to ours. Over 5,000 genes in the C. elegans genome
correspond to human genes, including important components of the nervous system.
Researchers can utilize this genetic information to “target” specific genes. For instance, by
eliminating a specific gene (known as targeted gene knockout), or by over-expressing a gene,
scientists can determine the function(s) of that gene and how it may be biologically important
for C. elegans. Neuroscientists can then test how the observed gene-function relationship applies
in mammals, including humans. Again, many genes serve the same function in C. elegans as they
do in humans, so we have learned a lot about neurobiology, and biology in general, from these
microscopic worms.

Reproduction in C. elegans

There are two sexes of adult C. elegans: hermaphrodites or males (there are no strictly females).
Hermaphrodites possess all the necessary components for self-fertilization and are the most
common (~50-99% depending on the environmental conditions). Hermaphrodite self-fertilization
results in genetically identical offspring, while male-hermaphrodite mating produces offspring
with greater genetic diversity. A single hermaphrodite worm can produce approximately 300

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OBSERVING BEHAVIOR: Learning and Memory with The Model Organism Caenorhabditis Elegans

offspring by self-fertilization or 1200-1400 offspring by mating with a male. A single adult male
can father about 3,000 offspring. See Figure 1 for a comparison of hermaphrodite and male C.
elegans.
Life Cycle
Whether an egg is self-fertilized with hermaphrodite sperm or fertilized via sexual reproduction
with male sperm, the resulting embryos hatch into the first larval stage (L1) approximately 14
hours post-fertilization (see Figure 1). Assuming favorable conditions, L1 larvae will develop into
L2 larvae. L2 larvae then continue to L3, L4, and finally, adulthood. Only 45-50 hours post
hatching, an adult hermaphrodite lays its first eggs, completing its reproductive life cycle. Adult
hermaphrodites continue to lay eggs for 3-4 days and then live for an additional 10-15 days.
However, if there is limited food, extreme temperatures, overcrowding or other environmental
stressors, animals will enter an alternate L3 stage called the dauer. In this dormant stage, worms
no longer feed, and locomotion is dramatically reduced.

A

B
Figure 1. Comparison between the C. elegans hermaphrodite and male.
A. C. elegans hermaphrodites undergo sexual or self-fertilization to produce embryos.

In approximately 14 hours, the embryos hatch into L1 larvae (A, below).
B. Male C. elegans. Note the lack of eggs (middle circle) and fanned tail (right circle) that distinguish the

male from a hermaphrodite. Adapted from WormAtlas.org

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Environmental influence on C. elegans

Organisms must be able to respond to stimuli they encounter as a means of survival. Therefore,
it is crucial to have a mechanism to sense and interact with the environment in an efficient
manner. C. elegans utilize chemosensation, sensing chemical stimuli that can signal everything
from food, danger, even the presence of other organisms. Sensing chemicals in the environment
can promote movement (which is called chemotaxis) towards attractive stimuli and away from
repellant stimuli. Think about how you respond to a noxious smell.

What about the smell of delicious food? Just as we respond appropriately to cues in our
environment, C. elegans respond to cues in their environment. For C. elegans, chemosensory
cues can also determine entry and exit to the dormant dauer stage of development. In this way,
the environment can have profound influence on behavior.
Learning and Memory in C. elegans

The field of learning and memory is primarily interested in understanding how the nervous
system acquires, stores, and recalls information from experiences. Organisms rely on their
previous experiences to maximize survival and positive outcomes, and avoid dangers in the
environment. Associative learning results from understanding the predictive relationship
between two stimuli (e.g. the bee sting and pain). Non-associative learning is when repeated
exposure to a stimulus results in a change in the strength of a behavioral response. While both
types of learning are important and regularly utilized, in today’s laboratory we will be focusing
on two types of non-associative learning: habituation and sensitization.

 Habituation is a process by which the repeated presentation of a stimulus leads to a reduction
in an innate behavioral response, as the organism learns there is no predictive information
contained in the stimulus.

 Sensitization, on the other hand, occurs when the repeated administration of a stimulus
progressively amplifies the innate behavioral response.

Studying Memory in the Laboratory

Although intuitive, ‘memory’ cannot be easily defined or directly measured. Rather, memory is
typically inferred from a change in an animal’s behavior. Therefore, our capacity to study memory
is conditional, not only on the animal being able to perform the task, but also on our ability to
detect a change in the performance. Such an indirect measure of memory can be problematic for
neuroscientists, because while conducting experiments they must differentiate between the
animal’s memory for an experience and its ability to perform the appropriate action. This is
known as the memory versus performance distinction.

To accurately determine the behavioral effects of a stimulus, like the formation of a memory,
researchers must do their best to make objective, unbiased observations and measurements of
the behavior. Several strategies can be used to achieve this, any of which may be employed in
this laboratory exercise.

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 To eliminate bias, some studies make various aspects of the experiment “blind” so that not
everyone knows what variables are being tested at a given point. For example, the
experimenter may not know the specific gene mutation in their experimental subjects.

 Another strategy is to gather multiple data points for the same item and then determine
statistical trends. This could mean that multiple individuals, each with his/her own biases and
perspectives score the same behaviors simultaneously, or that multiple subjects are used in
the study.

 A third strategy relies on a clearly defined set of criteria for scoring behaviors so that, for
example, different degrees of a behavior can be distinguished systematically.

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PRECAUTIONS FOR USING THE MICROSCOPE

You will be using a Nikon compound microscopes that many students used earlier this semester.
If you did not participate in a Microscopy lab previously, or you did and just need a refresher, follow
these instructions for a PRE-LAB ASSIGNMENT:

PRE-LAB ASSIGNMENT: Microscopy can be a challenging experience for the beginner as well as
the professional and only time and patience, with much practice get one close to perfect. Since
you will not have the luxury of time to practice fine tuning this skill, it is highly recommended
that you learn how to efficiently use a microscope before your lab session. This “Pre-Lab
Assignment” is designed to help you achieve a visual introduction to microscopy accompanied by
a simulated experience. Therefore, before taking the Quiz, you should:

 Watch YouTube video: “How to Use and Care for a Microscope” (4:45).
From NC Community Colleges: BioNetwork. Published Jun 28, 2017.
Available at https://www.youtube.com/watch?v=ROsc-IrJJ6M
o Only up to 2:48 will apply to this lab, but the whole video has useful information.
o One quiz question will relate to this video.

 Explore BioNetworks’ 3D Microscope simulation: “Virtual Microscope” ©2018.
Available at http://www.ncbionetwork.org/iet/microscope/
“INTRODUCTION - The Virtual Microscope provides a self-guided exploration of a common
laboratory microscope, its care and usage, and general information about the equipment
and its components. Through various activities you will have the opportunity to interact,
explore, and test your knowledge with this interactive application.”
o There are 5 tabs to experience: GUIDE, LEARN, EXPLORE, TEST, OPTIONS
o One Quiz question will relate to this simulation.

It is very important to carefully review the operational guidelines given below:

 ALWAYS read the instructions thoroughly prior to starting any of the experiments.
Be certain you and your lab partner are clear on your roles and the purpose of every
experiment.

 Always wear gloves, and always handle the dish by the edges.

 The microscopes you will be using have two lenses and are called binocular. You should
always adjust the eyepieces so that you can comfortably view the samples with both eyes
simultaneously. This can be tricky at first – try moving the eye pieces wider than your eyes,
then move the lenses together slowly until you can see through both with both your eyes.

 Using the microscopes at the 4X magnification setting may be optimal when working with
the dishes. The eyepiece magnification is 10X making the total magnification 40X on the 4X
setting.

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Brain & Behavior: CORE-UA 306

LABORATORY COVER SHEET
Name: _____________________________ Lab Partner’s Name: _________________________
Date of Lab: ___________________________ Date Due: _______________________________
Lab Instructor: _____________________________ Lab Section Number: __________________

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PROCEDURES and EXERCISES

 PART I: Environmental Influence on C. elegans Behavior

In this exercise you will examine how C. elegans respond to different stimuli in the
environment, utilizing the chemosensory system to go towards chemoattractants, avoid
chemorepellants, or ignore neutral signals.

You will observe the C. elegans through a compound light microscope. Place your plate on the
stage, and then use the coarse focus to bring the plate into view. If necessary, you can adjust the
eyepieces to accommodate the distance between the observer’s eyes. Turn the knob until the
organism comes into focus. Attempt this on your own first, and then ask your laboratory
instructor for help if you are struggling.
It is important to note that C. elegans can be fast moving and tend to burrow into the agar,
making it a challenge to count them or track their movements. Recall from the Microscopy lab,
that by using fine focus, you can observe specimens at different depths. Therefore, try to keep
your focus on the surface of the agar while observing the worms.

Procedures

For Part I, you will need:
- four agar petri dishes
- filter paper disks
- fine forceps (used to dip filter paper disks into each solution below)
- 3 Test Solutions labeled A+, B+, and C+
- 3 Control solutions labeled A-, B-, and C-

This experiment will be completed ‘blind,’ meaning you will not know the actual identity of
each solution while conducting the experiment.

YOU MUST USE GLOVES AT ALL TIMES DURING TODAY’S EXPERIMENTS.

ENSURE YOUR AGAR PLATES ARE REMOVED FROM THE MICROSCOPE LIGHT
SOURCE IN BETWEEN TRIALS TO PROVENT DRYING.

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[Steps 1, 2, and 3 may have already been done for you by the laboratory staff. If so, skip those
steps and begin with Step 4. If your plates do not have markings, start from step 1.]

1) Using a marker, draw two intersecting lines on the bottom of three of your plates, in order
to form 4 quadrants on each plate (i.e. on the agar side of the plate; not the cover. See
Figure 2).

2) Label a pair of “Test quadrants” across from each other “A+” on one plate, “B+” on the
second plate and “C+” on the third plate (Figure 2).

3) Label the other pair of “Control quadrants” on each plate as “A-“, “B-“, and “C-“ (Figure 2).

4) Using the forceps, dip one filter paper disk into “Test Solution A+”. Place the disk on the
agar, in the center of the quadrant labeled “A+”. Repeat this process for the second “A+”
quadrant.

5) Rinse the forceps. Repeat Step 4 with “Control solution A-”, placing each disk in the center
of the remaining control quadrants. Try not to create a liquid puddle on the agar. If you do,
soak up excess liquid with the tip of a Kimwipe® tissue.

6) Repeat Steps 4 and 5 for Plates B and C using the respective Test and Control solutions.

7) After your plates are set up, the lab instructor will dispense a 5-µL buffer solution
containing C. elegans onto the center of your plates, at the intersection.

Figure 2. Example of petri dish labeling. A+
Shown for Test Solution “A+” and Control solution “A-“ A- A-

IT IS IMPORTANT TO AVOID CROSS CONTAMINATION OF THE SOLUTIONS:
 Use squeeze bottle of water to rinse forceps between each transfer
 Kimwipe® tissues can be used to blot excess water from forceps.
 A waste beaker has also been provided.

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QUESTION 1:
Write hypotheses to explain what behaviors you would expect to observe from the worms if
any of the solutions are (A) a chemoattractant (B) a chemorepellant, or (C) a neutral solution.
A. Chemoattractant:

B. Chemorepellant:

C. Neutral:

8) Once the worms in buffer have been added to your agar plate, make an Initial
observation of them to obtain an approximate number and record it in the first column
of DATA TABLE 1. Then, allow the C. elegans to acclimate and move around for 25-30
minutes. While you are waiting, proceed to Part II of the laboratory experiment.

9) After at least 25-30 minutes….
For each of the 3 plates and for each quadrant, determine the approximate number of
worms located:
(i) in the middle of the plate, represented by the dashed circled area in the center,
(ii) in the surrounding area of each filter paper disk in each quadrant condition
A+

A- A-

Each partner should count the plates separately. Settle on a strategy so your counting is
unbiased. Complete DATA TABLE 1 with your observations.

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DATA TABLE 1. Observation of Worm Distribution* after 25-30 minutes.

Approximate # Approximate # # of worms near each # of worms near each
worms in middle worms in middle Test Solution disk control solution disk
of plate (Initial) of plate (Final)
1 2 Total
1 2 Total
Plate A

Plate B

Plate C

*Number of worms should be a qualitative approximation, not an exact number.

QUESTION 2:
Compare the distribution of worms for each of the plates. Did they show a preference for, or an
aversion to, any of the solutions?

QUESTION 3:
Re-evaluate your hypotheses to determine how your observations fit with your hypotheses.
How would you classify each of the Test and Control solutions? Use terms chemoattractant,
chemorepellant, or neutral to categorize each solution.

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QUESTION 4:
Why is it important that the worms respond to the solutions in different ways?

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 PART II: Learning and Memory

Experiment 2: Habituation versus Sensitization
In this experiment, you will work in pairs to study non-associative learning in C. elegans. Recall
that habituation is a process in which an initial behavioral response is diminished with training as
the organism learns (i.e. forms a memory) that a stimulus is not predictive. This contrasts with
sensitization, in which repeated exposure to a stimulus augments the behavioral response.
In today’s exercise, training trials will consist of observing the worm’s initial behavior, delivering
a tap stimulus, and recording any changes in the worm’s behavior following the tap. One partner
(the Trainer) will deliver the tap stimulus and observe the worm’s behavior, while the other
partner (the Recorder) runs the stopwatch, announces when to deliver the tap, and records their
partner’s observations. Through your observations, you will determine if the worm experiences
habituation or sensitization in response to this training protocol.

Procedure

 Label the bottom of your Training plate as “Part 2.”
 Remove the lid and draw a dot on the side of the agar plate to serve as a “target spot”

(Figure 3).
 Your lab instructor will inspect your set up before dispensing 1-2 L of buffer solution

containing C. elegans onto the plate. Keep the lid on your Training plate during the
training protocol.

Target dot

Agar Lid, off to the side

Figure 3. Training plate setup.
(Side view) Target dot placement on side of agar dish, without the lid.

1) Getting Familiar with C. elegans:

With your plate secured on the microscope stage, use Figure 1 and the hints below to help you
identify these differences in your worms:

- Eggs: small, ovular spheres, not moving

- L1 state: most recently hatched and smallest larvae

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- Adult hermaphrodite: largest worms, embryos inside, whip-like tail, move more slowly
than the males

- Male: slightly thinner and smaller body than the hermaphrodite, fan shaped tail, move
rapidly and tend to stroke other worms by doubling back on themselves

 Try to choose just one worm to observe from this sample.

 In response to each tap, the worms should show reversal swimming – movement in the
opposite direction of the baseline movement – following the tap.

 See if you can notice if all the worms respond in the same way as the one you are trying to
specifically observe.

To have a standardized set of scoring criteria, worms’ movements should be observed as the
following:

 Reversal – worm moves in the opposite direction compared to the pre-tap movement

 Movement – worm is in motion in the same direction compared to the pre-tap movement

 Inactive – worm is not in motion in any direction

2) Baseline Trials: First, you will observe baseline behavior and record how the worms behave
under normal conditions.

 The Trainer will bring an adult worm into focus and report the behaviors demonstrated
by the worm (Reversal, Movement or Inactive), while the Recorder will tally how many
times the worm engaged in each of the behaviors.

 For 5 trials of 10 seconds each, observe worm behavior WITHOUT any tapping (i.e. no
training).

DATA TABLE 2. Observed Behaviors for Baseline Trials

Trial Reversal Movement Inactive
1
2
3
4
5

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QUESTION 5:
A. What type of behavior do you predict the worms will show following the first training tap?
B. Following the 20th tap?
C. Write hypotheses to explain your predictions if the worm experiences habituation.
D. What about if the worm experiences sensitization?

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3) Training Trials: You will complete a total of 20 trials.

 Each trial will consist of the following steps delivered by the Trainer:
a) Stabilize the plate with your hand
b) Tap the side of the plate

 Trainer will deliver a tap every 10 seconds, carefully observing and reporting the worm’s
behavioral response to the Recorder.

 If you lose track of your worm, you can switch your observation to another worm; just make
sure you are recording behavior from a worm.

DATA TABLE 3. Observed Behaviors for Training Trials

Trial Reversal Movement Inactive
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

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4) After the Training trials, start your stopwatch to count forward (or set your timer for 15-30
minutes) in preparation to test memory. While you are waiting, answer the following:

QUESTION 6:
A. Do you notice any changes in behavior across the 20 training trials?
B. How does behavior on Trial 1 compare to behavior on Trial 20?
C. Was the change consistent with sensitization or habituation, or is it too inconclusive?

QUESTION 7:
Write a hypothesis explaining your predictions for the worm’s behavior when tested 15-30
minutes later, on the memory test.

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5) Testing Memory: Check your timer. If 15 minutes or more have passed, record the time that
has passed since you finished training and note it below in DATA TABLE 4 (should be 15-30
minutes).

 Like the 20 Training Trials, to test memory, you will deliver a tap every 10 seconds for 10 tests,
calling out behavioral observations for your partner to record.

 Each lab partner should observe the same size worm, large or small, that they observed
during the training trials.

DATA TABLE 4. Observed Behaviors for Memory Test
Test Reversal Movement Inactive
1
2
3
4
5
6
7
8
9
10
Time elapsed since training: ________

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QUESTION 8:
To understand how behavior changed across the baseline, training and memory tests, calculate
averages for behavioral performance: sum the number of times a worm engaged in each behavior
for every trial and divide it by the total number of trials.

For the training trials and the memory test you will calculate averages in bins of 5 trials (e.g. sum
of behavior on Trials 1 + 2 + 3 + 4 + 5/ 5 total trials) rather than all trials together, so you can tell
how performance changes. Fill in the calculated averages in the charts below.

DATA TABLE 5. Quantifying Observed Learning and Memory in C. elegans

Reversal Total Number for Baseline Trials Inactive
Movement

Total Number for Training Trials Averages for Memory Test
Trial Reversal Movement Inactive Trial Reversal Movement Inactive
1-5 1-5
6-10
11-15 6-10
16-20

QUESTION 9:
How does the behavior you observed during training compare to the behaviors during the test
completed 15-30 minutes later? Are your observations consistent with your hypothesis for
habituation or sensitization?

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QUESTION 10:
What advantage or disadvantage could reversal responses confer to C. elegans in their natural
environment?

QUESTION 11:
A. What advantage or disadvantage would habituation of this response confer to C. elegans in
their natural environment?
B. What about sensitization?

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CLEAN-UP PROCEDURES

When you are done with your work, you must clean up. You will not be permitted to leave the
lab until the lab instructor checks your lab bench.

 Turn off your microscopes and return the turrets to the 4X objective position.
 Discard only the used plates in the desktop biohazard waste bag; once filled, twisty-tie to

close bag and discard in brown biohazard box.
 Discard napkins and gloves in the grey trash.
 Please leave your lab station as clean and orderly as when you arrived.
 Before leaving the lab, double-check your area for your personal belongings.

THE LAB STAFF IS NOT RESPONSIBLE FOR ANY LOST OR STOLEN ITEMS.
Failure to properly clean up will result in a 5-point deduction from your laboratory assignment.

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