Brain and Behavior Student Manual 2022

Recording Action Potentials from the Model Organism Periplaneta Americana (L.)

 PART III: NEURAL SUMMATION

We will next explore neural summation, much like when we simulated summation of local,
graded potentials in the Model Dendrite lab. However, remember that while we modeled
intracellular recordings in the Model Dendrite lab, today we are using extracellular recordings.
Refer to the introduction of this lab to remind yourself of the differences between these
techniques.

1. Use your receptive field map to choose two spines that elicit a medium frequency of
activity in the nerve. Pick two spines that are far enough apart so that it will be easy to
separately, but simultaneously, stimulate both.

QUESTION 10:
Write a hypothesis predicting what will happen when you deflect both spines simultaneously.
Remember, there are two variables that can change when you are doing extracellular recording
of a nerve: spike frequency and root mean square. Be sure to comment on both.

2. Using the metal probes, deflect one of the spines you chose. Transfer and quantify the
data. Record the approximate frequency of spikes per second (Hz) as well as the RMS in
the table below. Repeat for the second spine you chose.

3. Now, using two metal probes, deflect both spines simultaneously. Again, transfer and
quantify the data as in #2.

Spines Deflected Frequency of Spikes Root Mean Square
(Hz) (RMS)
“A” (label this spine on your sensory map)
“B” (label this spine on your sensory map)
“A” and “B” simultaneously

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QUESTION 11:
When you stimulated A and B together, did you notice a change in the:
Frequency? RMS? Both? Or neither?

QUESTION 12:
Does your data support your hypothesis? Why or why not?

QUESTION 13:
Suppose you could precisely stimulate the cockroach leg through an embedded electrode.
A. Define what a ‘threshold’ amount of stimulus would equate to if we are measuring neural
activity using extracellular recording of the cockroach leg.
B. Describe what steps you would take to experimentally determine threshold.

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QUESTION 14:
Describe the similarities and differences between recording the summation of local, graded
potentials and the ‘summation’ of action potentials in the nerves. Be sure to include what
recording technique you would use to record these phenomena (e.g. intra- vs. extracellular) and
what summation means functionally for neural communication in each case.

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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.
 Do not save any data on your computer; close the Spike Recorder program upon completion.
 Do not remove the cockroach leg; Lab Staff will handle replacing the leg for the next class

section.
 Please leave your lab station and all experiment materials 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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ORGANIZATION OF THE BRAIN I: Microscopy – Neuron to Brain

LABORATORY 5
ORGANIZATION OF THE BRAIN I:

Microscopy – Neuron to Brain

SUMMARY
The aim of this lab is to extend the inspection of brain tissue to the level of the brain cell, or
neuron. As neuroscientists, we want to study how behaviors arise and are produced. To do this,
we need to quantify the behavior as well as examine the connectivity and gross structures of the
brain. For a more in depth analysis, we turn to individual neurons and how they transmit and
receive signals. In this lab, we will utilize a microscope to evaluate slides that have been mounted
with tissue from the visual cortex, coronal sections of the brain, and the cerebellum.
OBJECTIVES

• Understand how to use microscopy and different methods of neuronal visualization as a
tool to study neuronal morphology and proteins.

• Appreciate the layered organization of cortical and cerebellar structures.
• Identify distinct neuronal cell types and be able to identify their dendrites, cell body,

and axons.

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INTRODUCTION

Principles of Microscopy

The unaided eye can detect objects only up to 0.1 mm (10-4 m) in diameter. Since cells are smaller
than the limit of our vision, their observation requires the use of a light microscope to extend the
range of our visual capabilities. It was Robert Hooke in the 17th century that first used
microscopes to recognize cells.
The ability to view cells depends on both the properties of the microscope and the preparation
of the biological sample. The quality of the optical image produced by a microscope depends on
two parameters: magnification and resolution. The magnification enlarges our view of the cell,
and the resolution enables us to accurately distinguish its details.

To successfully use microscopy as a tool, it is also important to understand various operational
parameters of the microscope you are working with, such as field of view, light intensity,
working distance, and focal plane.

In today’s lab, you will be working with a compound microscope for the lab exercises (Figure 1).

Magnification

The construction and operation of a microscope relies on the properties of light. The
magnification achieved by a microscope arises from the ability of a lens to bend and focus light
to form an image that is larger than the original object being viewed. In the process of producing
the magnification, the lens inverts the image, much like looking in a mirror.

Modern light microscopes are compound microscopes. In these microscopes, magnification of
samples relies on two lenses, the objective lens and the ocular lens, which act together.

Figure 1 shows the organization of the optical components in a compound microscope. A light
source in the base of the microscope is provided to illuminate the sample. Light from the sample
enters the objective lens, which can be of varying magnification. At higher magnifications, the
objective lens will be closer to the sample. The image of the sample that is formed by the
objective lens is further magnified by the ocular lens, which is contained in the eyepiece and
focuses the image into the eye.

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Figure 1. The Nikon Eclipse E200 Compound Microscope.
[Image retrieved from http://www.nikon.com/products/microscope-solutions/lineup/upright_clinical/e200/]

It is important to remember:
Objective lenses and ocular lenses act together to produce total magnification of the specimen
slide being observed on your compound microscope, and is calculated using the equation below:
Total Magnification = Magnification of Objective lens x Magnification of Ocular lens

Resolution

Magnification is not the only important optical property of a microscope. Simply obtaining a
magnified image is useless unless the image is of sufficiently high quality to enable an observer
to distinguish important features. Resolution is the ability of a lens to distinguish between small
objects that are close together. Although we will not describe the resolution of a microscope in
detail, it is worth noting that it depends on the wavelength of light. Higher resolution is obtained
with shorter wavelengths, which is why a blue filter is often placed over the light source to
illuminate the slide.

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Operational Parameters

The magnification from the objective lens has important implications on the properties and
operations of a microscope. When moving from lower to higher magnification:
- diameter of the field of view will decrease (zooming in on a smaller section of the sample),
- light intensity will decrease (the light source becomes focused on the smaller field of view),
- and the working distance, or the distance between the slide and the objective, will decrease.
These relationships are summarized in Figure 2.

Figure 2: As the magnification of the objective lens increases, the working distance and the diameter of
the field of view will decrease.

Focal Plane

Like the eye, a microscope has only a limited depth of focus. Since all samples on a slide—even
tissue sliced very thin—are three-dimensional objects, only part of a specimen is in focus at any
given position of the objective lens. What you observe when looking through the microscope is
only one layer of the slide, which has a specific thickness called the focal plane. When using the
microscope, it is therefore important to make constant use of the fine focus adjustment,
especially when viewing a sample using the high-power objective lens.
To familiarize yourself with the Nikon compound microscope that you will be using in today’s lab,
study the labeled diagram in Figure 1. Note the position of the ocular lens, the turret containing
the objective lenses, and the location of the stage that is used to hold the slide.

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Microscopy as a Tool for Studying Brain Tissue

Just as the anatomical structures that make up the brain can give us information about function,
the microscopic structure of neurons can give us information about how neurons communicate
and coordinate all the complex behaviors we witness day to day. The basic features of a neuron
are the cell body, the dendrites, and the axon.

The cell body, or soma, is the metabolic center of the neuron and contains the nucleus, which
holds DNA. The nucleus is the only site in the neuron in which transcription, or the synthesis of
new copies of DNA, called mRNA, can take place.

The dendrites will appear as short processes emanating from the cell body. These structures are
the input region for neuronal communication and receive most of the synaptic contacts from
other neurons.

The axon is the long process that projects away from the cell body. Action potentials travel down
the axon to the axon terminal, where they trigger neurotransmitter release from the pre-synaptic
cell (which can then bind to receptors on the dendrites of the post-synaptic cell).

As you may have noticed in the sheep brain dissection, individual neurons are not visible to the
naked eye, which is why microscopy is such an important and useful tool. To visualize cells we
used various tricks to ‘stain’ or highlight different components of the neuron. In today’s lab, you
will use slides that have been treated with three different stains. Choosing a stain depends on
what you are interested in visualizing; some stains will label whole neurons including all their
delicate processes, while other stains that will label only a specific protein at synapses or another
area of the neuron. You can also choose probes that will stain only one kind of neuron, such as
the inhibitory GABA-ergic neurons, while leaving all other neuron types unstained.

Today you will look at prepared slides of each of the following:
• Visual Cortex, Nissl stain
• Coronal Sections, Golgi stain
• Cerebellum Sections, Silver Stained

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Layered Structure of the Visual Cortex

Many brain structures have an intricate internal organization of substructures, like the thalamus,
which is made up of multiple discrete nuclei. The cortex, a 1-2 mm thick layer of cells at the dorsal
surface of the brain, also has internal organization. Close inspection of the cortex shows that it is
not just a homogeneous collection of neurons, but it is itself precisely layered (see Figure 3
Handout).

In the cortex, layers of varying densities of neurons and cell types can be seen above a layer of
axons, which constitutes the white matter. In general, the cortex contains six layers, numbered
1 to 6 from the surface to the white matter. The exact number of layers differs from area to area
and among species. As you will discover in the exercises, each layer can serve a different
functional purpose, and any developmental disruption in creating this organization can be
catastrophic.

Figure 3 [HANDOUT].
Layers of primate visual cortex,
stained for Nissl substance.

The cortical slide you will look at is a Nissl stained visual cortex. The Nissl stain is a substance that
binds to negatively charged nucleic acids, like DNA and mRNA, which are highly localized in the
cell body. So, dark Nissl staining will indicate a high density of neuronal cell bodies.
Review the visual cortex layers in Table 1 along with Figure 4-Handout to gain an appreciation
for the intricacy of this organization.

Table 1. Layers of the Visual Cortex

Layer Cell types and Description

I Mostly dendrites - collects inputs; networking area

II Somata and dendrites of pyramidal cells; many axons and dendrites from other layers

III-A Somata and dendrites of pyramidal cells; many axons and dendrites from other layers

III-B Somata and dendrites of pyramidal cells; many axons and dendrites from other layers;

High thalamic and IV-C input

IV-A Granule-like cells; dark band; lacks pyramidal cells

IV-B Low density pyramidal cells; strong input from IV-C, conveys input to M pathway/dorsal stream

IV-C Innervates IV-B and III-B

V-A Innvervates all layers except IV-B and IV-C beta

V-B Sends recurrent axons to III-A

VI Sends recurrent axons to IV-C beta; receives LGN projections and projects back to LGN

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Individual Neurons in Coronal Sections

As was already mentioned, there are many different kinds of neurons, some having completely
different morphologies, or shapes. The primary class of neurons that sends excitatory messages
(known as EPSPs) are glutamatergic neurons, many of which are classified as ‘pyramidal’ neurons.
These neurons are aptly named for their triangular cell bodies (Figure 4A). The primary class of
neurons that sends inhibitory messages (known as IPSPs) are GABAergic neurons, many of which
are classified as ‘inhibitory interneurons’ (Figure 4B).

A.

Axon B.

Cell body
Dendrites

B.

Figure 4: Pyramidal Neuron.
A. Golgi stain (20X) of a cortical pyramidal neuron.
B. Golgi stain (20X) of a cortical interneuron.
Images courtesy of Mike Hawken.

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The beauty (and mystery) of the Golgi stain process is that some neurons take up the stain and
transport it throughout the extent of the cell, while some neurons do not take up the dye at all
and remain unstained. Those neurons that stain, do so completely, including the finest branches
of their dendrites, and stand out in brown or black against a clear background. Occasional glial
cells such as astrocytes are similarly stained.
An important feature of this method is that it stains only a small proportion of the cells in the
sample, allowing researchers to see the morphology of individual neurons. If all cells were
blackened, it would be impossible to resolve the structural details of individual cells in the dense
forest of neurites (dendrites and axons).

Cell Layers in the Cerebellum

The cerebellum is an evolutionarily conserved structure that has a highly convoluted surface.
Because of the extensive folding of the cerebellar surface in the form of thin, transverse folia,
85% of the cortical surface is concealed. Therefore, there is a large cortical area distributed over
a small amount of space. The cerebellar surface is about three-quarters as extensive as that of
the cerebral cortex. Like the cortex (including the visual cortex which you will see today), the
cerebellum is organized into layers. Figure 5 depicts the three cortical layers that can be seen.
From the surface to the white matter of the folium, the layers are:
(1) the Molecular layer, (2) the Purkinje cell layer, and (3) the Granule cell layer.

Purkinje cell layer

Figure 5: The folia of the cerebellum. Note the Purkinje, molecular, and granule cell layers.
Image retrieved from http://www.siumed.edu/~dking2/ssb/NM030b.htm.

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The cerebellum samples you will use in today’s lab are treated with a silver staining method.
Reduced silver methods produce dark deposits of colloidal silver in various structures, notably
the protein-containing filaments inside axons. The most widely used techniques of silver staining
are those developed by Ramón y Cajal, one of the pioneers of the study of Neuroscience.

PRECAUTIONS FOR USING THE MICROSCOPE

NOTE: MICROSCOPES ARE DELICATE AND EXPENSIVE INSTRUMENTS.
PLEASE USE THEM CAREFULLY.

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

1. The Nikon microscopes are used by many students each semester.
It is a good practice to clean the ocular lenses with lens paper before you begin using a
microscope.

2. When inserting or removing slides, turn the turret to the lowest (“smallest”) power objective;
this will avoid damaging the objective lens. When turning the turret, do not touch the
objective lens casing; instead, use the black ring at the top of the turret.

3. Always examine a slide first using the lowest power objective.
Focus on the slide before moving to the next magnification, and repeat this process until you
have reached the highest magnification.

4. NEVER use the coarse focus adjustment when viewing a slide with a high power objective
(10X or 40X). Only use the fine focus. The distance between the lens and slide is very small
and you may crack both the slide and the lens.

5. Always handle slides by the label or the edges. Do not put your fingers over the biological
sample since your fingerprints will degrade the image.

6. When returning slides to the slide box, take extra care to place them straight in the slide
holders, and not askew, to prevent the slides from chipping or breaking.

NO 1
YES 2
3
4
5
6
7
8

7. 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.

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8. 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.

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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: FAMILIARIZATION WITH THE MICROSCOPE

A. Magnification

QUESTION 1:
A. What magnification is stamped on the housing of the ocular lens?
B. What is the magnification of each objective lens?

QUESTION 2:
What is the total magnification achieved by the microscope using each of these objectives?
Show all calculations in the space provided.

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B. Image Orientation

Ensure the turret is turned to the 4x objective and insert the slide containing newsprint. The slide
should be oriented so that the letters are upright (oriented as for normal reading).

- Adjust the stage until the letters on the slide are illuminated by the lamp.
- Use the coarse and fine focus adjustments to obtain a focused view of the slide.
- Use the stage adjustments to move the slide left to right then toward and away from you;

be sure not to push the stage around with your hands
- While adjusting the position of the stage, look at the slide directly and also through the

microscope.
QUESTION 3:
A. Draw the letters as viewed directly on the slide and as viewed through the microscope.
Use the spaces provided under your sketches and on the next page to answer the following
questions:
B. What is the orientation of the letters as viewed through the microscope? Note both the top-
to-bottom and side-to-side orientation as compared to viewing the letters directly.
C. How does moving the slide to the right on the stage affect its movement as viewed through
the microscope? Similarly, how does moving the slide towards you affect its movement as viewed
through the microscope? Use a sketch or diagram to illustrate the different movements.
A.

Newsprint slide

Total Magnification = 40X
B.

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Answer for QUESTION 3C.

C. Focal Plane

As discussed in the introduction, each slide is in fact a three-dimensional object. This exercise will
illustrate how the focal plane is important for viewing samples. You will use a slide containing
three threads of different colors, crossed over one another.
- Turn the turret to the 4x objective and place the slide containing colored threads on the stage.
- Focus on the threads with the 4x objective, and then the 10x objective.
- Using the coarse focus, move the knob back and forth very slowly. This will enable you to

focus on each individual colored thread.
- By noting which thread is in focus at a specific position of the objective lens, you can deduce

the depth arrangement of the threads.
QUESTION 4:
As you adjusted the coarse focus, write a description of what you observed when viewing the
threads? From “bottom to top,” in what order are the threads stacked?

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As a reminder….

Please be very careful with the slides and microscope objectives, as they are very expensive.
When moving to higher magnification using the objective lenses, take care to move the objective
lens towards the slide carefully and do not bring the objective lens into contact with the slide.
When using the high-power objectives, use the fine focus only.

PART II: INSPECTION OF NISSL-STAINED VISUAL CORTEX

Set the magnification to a point at which you can see the sample and use the fine focus to adjust
the image to your eyes. You should be able to see the layers of the cortex clearly.
QUESTION 5:
Use the space below to (A) make a drawing of the visual cortex layers and (B) label each transition
in the density of staining which will correspond to different layers. You can refer back to the
Handout for reference. Note the Total Magnification of your observation under your sketch.

Total magnification: ___________

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PART III: INSPECTION OF GOLGI-STAINED CORONAL SLICES

Begin by looking at the sample under the lowest magnification. Use the fine focus to bring several
neurons into focus. Remember, a slice is a 3D object, and not all neurons will be in focus at the
same time.
QUESTION 6:
Make a sketch of the slice under 4X magnification.

-------------------------------------------------------------------------------------------------------------------------------
Now, switch to 10X magnification. Again, bring the image into focus using the fine focus.
You should now be able to see at least two distinct types of neurons.
QUESTION 7:
A. Make a detailed sketch of two neurons that appear to be distinct cell types, meaning their
morphology is noticeably different.
B. For each, label the dendrites, cell body, and (if visible) the axon.

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QUESTION 8:
Any brain region can have dozens of distinct neuronal and glial populations (e.g. astrocytes)
populations. Hypothesize why having so many different kinds of cell types could be useful for
brain function.

QUESTION 9:
One method to visualize different cell types is to use different markers that stain specific classes
of proteins. GFAP is a common protein used to identify a certain cell type.
Look up and record (A) what GFAP is, and (B) hypothesize what cell type would be stained if you
used a GFAP-specific probe.

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PART IV: INSPECTION OF SILVER-STAINED CEREBELLAR SLICES

You will be examining the Molecular, Purkinje, and Granule cell layer in cerebellar folia.
Start by viewing the slide at 4X magnification, and then move to 10X. Use the fine focus to bring
the 3 layers into view.
QUESTION 10:
What are the primary functions of a Purkinje cell?

QUESTION 11:
A. Make a detailed sketch of the image you see at 10X magnification.
B. Label the three cell layers as well as the dendrites and cell body of a Purkinje cell.

QUESTION 12:
A. How many cell bodies thick is the Purkinje cell layer?
B. Which layer do the Purkinje cell dendrites branch into?

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CONCLUSION QUESTIONS

After completion of the lab, you should be able to answer these conclusion questions at home.
QUESTION 13:
In the visual cortex and cerebellum samples, you should be able to see a distinctive multi-layered
organization, which is common to the cortex throughout the brain.
A. Hypothesize what this organization could contribute to brain function.
B. What advantages are there to having these layers?

In the cerebellum samples, you saw the cell bodies of Purkinje cells. Although you may not have
seen it on the slide, cerebellar Purkinje cells have one of the most elaborate dendritic arbors, or
processes, in the brain.Below is an image of a typical Purkinje cell. Compare this image to the
neurons you drew from the Golgi stained coronal sections.

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QUESTION 14:
What do the differences in dendritic architecture mean functionally for neural communication?

QUESTION 15:
Parkinson’s disease results in a loss of a very specific kind of neuron in the brain region known as
the striatum – dopaminergic neurons, or neurons that produce the neurotransmitter dopamine.
A. How could you use microscopy and the other tools discussed in today’s lab to study dopamine
neurons in healthy brains and brains affected with Parkinson’s?
B. What would you expect to see?

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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 the microscope lamp and return the turret to the 4X objective position.
 Clean all slides with lens tissue and return them to your assigned slide box; you should

have 5 slides in your box, per the provided slide list.
 Follow your instructor’s directions on how to clean your microscope station.
 Discard used lens tissue (from the waste bin on your bench) into the large trash can and

return empty waste bin to your 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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BRAIN ANATOMY AND INNER STRUCTURES: Dissecting the Sheep Brain, Part II

LABORATORY 6

BRAIN ANATOMY AND INNER STRUCTURES:
Dissecting the Sheep Brain, Part ll

Note: You will be taking pictures of your sheep brain dissections in this lab. We suggest you
bring your own camera or camera phone.

SUMMARY
The brain is the principal organ of behavior. First we will give you some instruction and guidance
about terms that are used to describe the structures of the brain that we know are important in
determining different types of behavior. This relationship between the specific parts of the brain,
the structure, and the specific behaviors that are controlled by brain structures, is called the
structure-function relationship. In this lab, you will look at the surface and internal structures of
the sheep brain. In the coming weeks, we will extend our examination of this question by building
a clay model of the human brain.
Two very useful resources to refer about sheep brain dissection before* and after this
laboratory session:
- An interactive sheep brain atlas provided by The University of Scranton Behavioral

Neuroscience Lab. http://www.scranton.edu/faculty/cannon/sheep/framerow.html.

- “Sheep Brain Dissection” narrated YouTube video by Dr. Aaron Ament, Instructor of
Anatomy & Physiology I and Supervisor of The Cadaver Laboratory at Minot State
University. https://www.youtube.com/watch?v=r9EGoYW7dPA
 There will be a quiz question regarding this video.

OBJECTIVES
 Be able to use terminology for neuroanatomical directions to describe locations
 Understand the anatomical organization of structures in the sheep brain and the
relationships between them
 Understand how function maps onto the structures you identify
 Make your own brain atlas using photographs taken during the lab session

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INTRODUCTION

 Macroscopic Inspection
Why are we using sheep brains? You’ll find that being able to look at and dissect a real brain it is
quite different from looking at diagrams and slides. Sheep brains are often used in teaching labs
because they are relatively large in size (so seeing neuroanatomical structures is easy), they show
many of the structures that are common to most mammalian brains, and they are readily
available.
`

 Anatomical Directions
Before collecting your sheep brain, it is important that you understand the terminology
conventionally used in neuroanatomy. Directions in the nervous system are usually described
relative to the neuraxis, an imaginary line drawn through the spinal cord up to the front of the
brain. An animal like an alligator has a straight neuraxis because a straight line can be drawn from
between the eyes continuing down the spinal cord (Figure 1). In this example, the front end is
anterior, and the tail end is posterior, these can also be referred to as rostral (toward the beak)
and caudal (toward the tail), respectively. The top of the head and the back are part of the dorsal
surface, while the ventral surface faces the ground.

These directions are more complicated in humans because we do not have a straight neuraxis.
Because we stand upright, our neuraxis bends so that the top of the head is perpendicular to the
back. In this case, the dorsal part of your brain is at the top of your head (not the back of the
head) and the ventral surface of your brain is above the bottom of your chin, perpendicular to
your neck (Figure 2). In humans, rostral is towards your face, and caudal is towards the back of
your head.

In both examples, medial is toward the midline of the body or brain, and lateral is toward the
side (away from the midline). The anatomical directions can be confusing, so ask questions if
you’re still uncertain about them.

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Figure 1: Animal Neuraxis. Figure 2: Human Neuraxis

 Dissection Terminology

Some structures you will only be able to see after cutting, or sectioning, the brain. Typically, there
are three planes in which a brain can be sliced: sagittal, coronal, or horizontal (Figure 3).

 A sagittal section is a vertical section that divides the brain into left and right portions.
As you’ll see later in the lab, a midsagittal section is a vertical section made down the center
of the brain, which divides the brain along the midline into two equal halves.

 A coronal section is a vertical section that divides the brain into front (rostral) and back
(caudal) portions.

 A horizontal section divides the brain into upper and lower portions

Figure 3: Schematic showing the three planes in which the brain can be sectioned.

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 Coarse Brain Structures

The cerebral cortex is the outermost layer of the brain, and is visible without any dissection.
The cortex is divided into two symmetrical halves, called hemispheres, that are separated by the
medial longitudinal fissure.

 Hemispheric Differences

Structurally, the right and left hemispheres appear nearly identical. Functionally, however, there
are some key differences between them. For example, two areas of the left hemisphere, Broca’s
and Wernicke’s areas, are believed to be specialized for language production and
comprehension, respectively. While these hemispheric differences tend to be over-exaggerated
in popular culture, it is the case that neuroscientists are constantly trying to understand how
complex behaviors can be localized to specific structures within the brain.

Important advances in understanding which brain areas are important for controlling different
behaviors came from studying patients with brain damage. In these cases, scientists attempt to
trace deficits in behavior or cognitive function back to the damaged regions of the brain.
For example, if there is damage to the left side of the cerebral cortex it can result in impaired or
lack of movement of the right side of the body (paralysis). We now know this is because the left
hemisphere controls movements on the right side of the body (and vice versa). In neuroanatomy,
we refer to structures on the opposite side of the body as contralateral connections. In contrast,
ipsilateral refers to connections on the same side of the body.

 Gray and White Matter

Like every other organ, the brain is made up of specialized cells called neurons. The cell bodies
and dendrites of neurons are closely packed together and on visual inspection they look gray (or
sometimes off-white). As such, this is called the gray matter of the brain. The axons that connect
neurons in one area of the brain with neurons in another area tend to look white, and are called
the white matter. The white areas have this color because the axons are covered with a fatty
coating called myelin. When you inspect the brain you will see regions that look gray and others
that clearly appear white.

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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

General Notes

You must wear gloves during this laboratory.
You will be provided a saline bottle. It is used to keep the sheep brain moist and to prevent the
sheep brain from drying out. Use it periodically throughout the lab.
You will be identifying and labeling structures in the sheep brain and taking photographs as you
work through each step.

This camera icon indicates when you should be taking photographs of the structures
you’ve labeled. You will then compile your photographs into a powerpoint template to
create your own atlas.
The Sheep Brain Atlas-Template_Part2_S22.pptx is available on Brightspace for download and
will also be provided during the lab session for students as a reference guide.
Each group will have a laminated copy of The Sheep Brain: A Basic Guide (by R.K. Cooley and C.H.
Vanderwolf) on their lab bench to help with the identification of key structures. Relevant pages
of the brain guide will be referenced throughout the lab.

IMPORTANT SAFETY NOTICE:
THE SHARP INSTRUMENTS USED IN THIS LAB MUST NOT BE MISHANDLED OR MISUSED IN ANY WAY.

ANY PERSON FOUND UTILIZING THESE TOOLS WRONGLY WILL RECEIVE A ZERO FOR THE LAB
ASSIGNMENT.

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 PART I: Surface Examination of the Sheep Brain

When you first get your sheep brain, DO NOT make any slices! Before cutting into the brain it is
important that you understand the directions of your sheep brain.

o Using the toothpicks and label tape, identify and label the rostral and caudal ends of the
brain by marking the dissection pan. Then identify and label the dorsal and ventral
surfaces dirently on the brain. Use the images in Figure 4 as a guide.

Anatomists have broadly defined four different zones of the cortex as the frontal, temporal,
parietal, and occipital lobes.

o Using the toothpicks and label tape, identify and label the four primary cortical lobes on
the sheep brain. Use the images in Figure 4 for reference. Have your lab instructor verify
that your labels are correct before moving on to the next step.
Take two photographs of the primary cortical lobes you’ve labeled:
a dorsal (top) view and a lateral (side) view.
Add photos to SLIDE 3 of your Sheep Brain Atlas-Template_F22.pptx

ROSTRAL

CAUDAL

Figure 4: Sheep brain showing the different brain lobes from dorsal and lateral views. Note that the posterior third
of the dorsal surface, the cerebellar cortex, is also wrinkled with gyri and sulci.

QUESTION 1:
Why do you think we talk about the brain as four separate lobes?

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The front, or anterior, two-thirds of the dorsal surface of the brain is not smooth, rather it is
highly convoluted. This is called the cerebral cortex. The bumps are called gyri (or, singular as a
gyrus), and the indentations are called sulci (sulcus – singular).

o Using the toothpicks and label tape, identify and label a gyrus and a sulcus (see page 6,
The Sheep Brain: A Basic Guide).

Take a photograph that clearly shows your labeled gryus and sulcus.
Add this photo to Atlas - SLIDE 4.

Similarly, just caudal to the cortex is a small, highly ridged structure known as the cerebellum (or
‘little brain’).

o Using the toothpicks and label tape, identify and label the cerebellum (see page 6, The
Sheep Brain: A Basic Guide).

Take a photograph that clearly shows your labeled cerebellum.
Add this photo to Atlas - SLIDE 4.

QUESTION 2:
Not all animals have sulci and gyri (for example, the surface of the rabbit brain is smooth).
Why do you think these features have evolved in some species?

Ventral View

Flip over your sheep brain to view the ventral side. You may see a thick material still attached to
the brain, known as the dura mater. The dura is one of the layers that encases and protects the
brain. If you see any dura, ask your lab instructor for help removing it to expose the ventral
surface.
Sensory and motor information is transferred between the brain and other regions of the body
via cranial nerves, small fibers that are most visible on the ventral side of the brain. In total there
are twelve pairs of cranial nerves, and each is related to a specific behavior or function. Often
when the dura is removed, the small cranial nerves are destroyed. However, you will still be able
to see the most prominent structures for relaying sensory information: the optic nerve and the
olfactory bulbs.

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Label and photograph the optic nerve and olfactory bulb. Using the color handout of the
ventral side of a sheep brain (Figure 5), try to identify and label other nerves that are still
intact on your sheep brain (also see page 10, The Sheep Brain: A Basic Guide). Table 1 lists
the cranial nerves and some examples of how each functions.
Add your photo to Atlas - SLIDE 5.

Figure 5. Handout of sheep brain, inferior view.Retrieved from
http://images.slideplayer.com/26/8315607/slides/slide_65.jpg

Table 1. Cranial nerves.

CRANIAL NERVE – CRANIAL FUNCTION
Name NERVE –
Number conveys smell information
Olfactory tract transmits visual information from the retina to the optic chiasm
I coordinates eye movements (dorsal, ventral, medial rectus, ventral oblique), pupil

Optic nerve II constriction
controls eye movements: superior oblique muscle, moves eye downwards and inwards
Oculomotor nerve III
general sensation from face, scalp, nasal, and oral cavities, muscles of mastication
Trochlear nerve IV movement of eye ball, lateral rectus muscle

Trigeminal nerve V sensation from anterior 2/3 of tongue, muscles of facial expression
hearing and balance
Abducens nerve VI
taste, swallowing, salivation
Facial nerve VII general sensation and control of pharynx, larynx, esophagus

Vestibulocochlear nerve VIII movement of head and shoulders
Glossopharyngeal nerve IX movement of tongue
X
Vagus nerve XI
Spinal accessory nerve XII

Hypoglossal nerve

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We will now examine the midbrain and brainstem structures from the ventral view.
Proceed caudally to the pons, a thick transverse band of fibers that arches across the ventral
surface of the brainstem. Trace the fibers of the pons laterally and observe that they extend into
the cerebellum.
Posterior to the pons lies the medulla, the most caudal portion of the brain stem. Caudal to the
medulla is the start of the spinal cord.

Label and photograph the pons, medulla, and spinal cord (see page 10, The Sheep Brain:
A Basic Guide). Add this photo to Atlas – SLIDE 6.
QUESTION 3:
There are marked differences that can be observed between the brains of different species.
However, the brain stem structures are consistently similar.
Look up the functions of the pons and medulla. In the space provided below, hypothesize why
these structures are highly conserved across species.

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 PART II: Parting the Hemispheres

Gently part, but do not tear, the two hemispheres to reveal the corpus callosum positioned inside
the medial longitudinal fissure. It may be easiest to first insert your thumb or a blunt tool to
loosen the two hemispheres, and then gently pull apart the two hemispheres (Figure 6). Deep
within the brain you should see a flat, white band in between both hemispheres: this is the corpus
callosum.

Label and photograph the exposed and uncut corpus callosum (see page 14, The Sheep
Brain: A Basic Guide).
Add this photo to Atlas – SLIDE 7.

A. B.
Figure 6: Apply pressure to start to pull apart the two hemispheres as in image A, using a blunt instrument or your
thumbs to loosen the hemispheres. Then, as in image B, gently pull apart the hemispheres to reveal the corpus
callosum.

QUESTION 4:
What is the purpose of the corpus callosum?
Why do you think this might be important for brain function?

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 Midsagittal Cut

We will begin our examination of the inner structures of the brain by cutting the brains mid-
sagittally, to fully split the two hemispheres apart.

o Use smooth, one-way motions to cut through the corpus callosum with the small knife.
Do not use sawing motions, as this will prevent observations of symmetry and intactness
of internal structures.

Focus on one hemisphere of the brain. Notice that the gyri and sulci of the cerebral cortex extend
deep into the median longitudinal fissure, and is still visible on the medial surface. Just ventral to
the cortex is a white band of fibers known as the corpus callosum. The corpus callosum forms
the roof of the open space known as the lateral ventricles (each hemisphere contains one of the
lateral ventricles, so you will only have one lateral ventricle in the hemisphere you’re working
with).
The flat, circular region ventral to the lateral ventricles is the thalamus. Notice how the area
ventral to the thalamus forms an almost teardrop shape: the area within this triangle is the
hypothalamus.

Identify, label and photograph the corpus callosum, lateral ventricle, thalamus and
hypothalamus (see page 14, The Sheep Brain: A Basic Guide).
Add this photo to Atlas – SLIDE 8.
QUESTION 5:
What material does the lateral ventricle contain? Explain the function of this material in your
answer.

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ORGANIZATION OF THE BRAIN II: Dissecting the Sheep Brain

 Internal Anatomy: Blunt Dissection of One Hemisphere

Place one of your hemispheres dorsal surface up. You will be dissecting down using your scalpel
to expose the dorsal surface of the corpus callosum and the strucures that lie underneath.

o Start by removing some of the cortex down to the corpus callosum. This is best done by
making one cut through the cortex perpendicular to the corpus callosum, and another
parallel to it in the natural space between the two structures (Figure 1 demonstrates the
cuts).

Figure 1:
To expose the internal anatomy,
start by making 1 cut perpendicular
to the corpus callosum, along the
dashed line, as seen in A.

A. B.

o Next, make a second cut just above the corpus callosum so you can peel away the cortex
above and expose the dorsal surface of the corpus callosum (along the dashed line in B).

Be careful as you make your cuts: work slowly since you do not want to cut through the corpus
callosum. You may want to use your thumb to help remove the tissue.** The goal is to be able to
view the medial and dorsal surfaces of the corpus callosum. Once you have the corpus callosum
exposed, notice the size and thickness of this structure.

o Now, gently cut away the corpus callosum so you peel it up, revealing the structures
underneath. You should notice two smooth, shiny white structures underneath.

The anterior structure is the caudate, while posterior structure is the hippocampus. These
structures dive down ventrally into the cortex, so from this view you are seeing only the dorsal
parts of these curved structures. In sheep, the hippocampus extends laterally and posteriorly. In
fact, this structure is named for its unique shape: hippocampus actually means ‘seahorse’ in latin.
When we model the human brain, you will notice a species specific difference in the shape of this
structure.

Label and photograph the exposed hippocampus and caudate.
Add this photo to Atlas – SLIDE 9.

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QUESTION 6:
Look up the functions attributed to both the hippocampus and caudate. Hypothesize what the
functional outcome would be if each of these structures were to be damaged.

o If you have time at the end of your dissection and exercises, you can continue to dissect
out the entire hippocampus. Are you able to notice the curved, “seahorse” shape?

 Anatomy of the Cerebellum

o Remove the cerebellum from the brain stem.
You should notice that, like the cerebral cortex, the surface of the cerebellum is also highly
convoluted. The cerebellum alone contains more neurons than the entire rest of the brain.

o Make several sagittal cuts through the cerebellum using your knife (refer back to the
beginning of this lab if you are unsure what a sagittal section means).

Notice the delicate, tree-like appearance, known as the arbor vitae, or ‘tree of life’.
Take a photo of your cerebellum sections, with a label identifying the arbor vitae (see
page 14, The Sheep Brain: A Basic Guide).
Add this photo to Atlas – SLIDE 10.

QUESTION 7:
Mr. X was in an accident that resulted in damage to his cerebellum. He has been diagnosed with
cerebellar ataxia. He tends to lose his balance easily, has trouble walking and has difficulty
focusing his eyes on features in his environment.
Based on Mr. X’s symptoms, what can we hypothesize is the function of the cerebellum in a
normal healthy adult?

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 Part III: Coronal Sections

You will now be examining the same structures but from a diferent perspective, this time making
coronal cuts (vertical slices) as indicated in Figure 2 below.

Figure 2: Locations of the three coronal cuts.

Locate and label the corpus callosum, thalamus, and hippocampus in all of the coronal
sections (see pages 18 and 20, The Sheep Brain: A Basic Guide). Note: Not all structures
are necessarily visible in each section.
Add photo to Atlas – SLIDE 11.
QUESTION 8:
Of the corpus callosum, thalamus, and hippocampus, which structure extends the most rostral?
Which extends most caudal?

Label and photograph an area of gray matter and an area of white matter in your coronal
sections.
Add photo to Atlas – SLIDE 12.

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QUESTION 9:
What is the function of the thalamus? Why do you think it needs to be so large?

QUESTION 10:
As a reminder, gray matter is made up of cell bodies, while white matter (like the corpus
callosum) is formed by axons. Conventionally we talk about the brain as disparate structures.
Why is it so important that we have a lot of white matter throughout the cortex?

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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.
Failure to properly clean up will result in a 5-point deduction from your laboratory assignment.

 SAFETY NOTE: When walking about the room with your scalpel, ALWAYS cover the blade with the
provided cork.

 Discard no more than 2 dissected sheep brains with pieces of brain tissue in the benchtop waste
bin. Close the bag tightly and place it in the large brown “Regulated Medical Waste” box. If extra
benchtop bags are needed, ask your Instructor. DO NOT THROW UNWRAPPED SHEEP BRAINS OR
BRAIN TISSUE INTO THE “REGULATED MEDICAL WASTE” BOX.

 Clean all instruments carefully in the sink and dry them with the paper towels provided to prevent
them from rusting; please return them to your bench clean and dry.

 DO NOT LEAVE PIECES OF BRAIN TISSUE IN THE SINK—PUT THEM IN THE RED PLASTIC BAG
RESERVED FOR THIS PURPOSE.

 Wash dissection pan with soap and water; return clean pan upside down to dry on lab bench near
sink.

 LABELS AND TOOTHPICKS go in the regular trash, NOT DOWN THE DRAIN!!
 When you have finished washing up, clean the surface of the bench using a squeeze-bottle of

ethanol and a paper towel.
 Please strive to 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.

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