ANAT 242 - NEUROBIOLOGY

Semester 2 - Otago University, Dunedin Campus.

Lectures
Mondays and Tuesdays 11:00 - 11:50 am, alternate Wednesdays 10:00 10:50 am
Colquhoun Lecture Theatre (Dunedin Hospital)
Mid-semester break: 30 August - 3 September

Practical classes
Mondays 2:00 - 3:50 pm or 4:00 - 5:50 pm or 6:00 - 7:50 pm
Histology Classroom (1st floor, Lindo Ferguson Building)

Course Coordinator
Please note: contact by email in the first instance.
Dr Ping Liu can be contacted via email: ping.liu@stonebow.otago.ac.nz.,
phone 479 7536, Room 406 LFB.
Lecturers
The lecturers for this paper in 2010 will be: Dr Ping Liu, Dr Ruth Napper
and Prof. Gareth Jones. The Teaching Fellow is Mr Brad Hurren
(bradley.hurren@anatomy.otago.ac.nz ). Laboratory demonstrators will be
available to assist you during the practical classes.

Course Objectives
The objective of ANAT 242 is to build on knowledge and understanding
acquired from the First Year Health Science programme (specifically,
HUBS191 & HUBS192) to provide a more detailed appreciation of the
principles of the human structure and understand in relation to the function
of the Nervous System. Studies undertaken during ANAT 242 will provide a
sound foundation for 300-level Neuroscience studies in this and other
Anatomy Departments. Specific learning objectives for this paper will be
provided at the start of semester 2 but you will be encouraged and indeed
expected to go beyond the mere accumulation of factual anatomical
information and begin to understand the processes by which this information
was obtained and the value that should be placed upon it.

Course Content
ANAT 242 will be delivered as three major modules directed at three
distinct levels: human anatomical structure of various systems (including:
skeletal, muscular, cardiovascular and the nervous system), cellular
organization and molecular events. The former will provide a reasonably
detailed and systematic overview of the anatomical organization of the brain,
peripheral nervous system and their related anatomical structures. This part
of the paper will make major use of laboratory teaching. The second module
concerning the cellular organization of the nervous system will consider the
specialized structure of a variety of cells which make up the nervous system.
The final module will look within neurons to begin to describe the molecular
events which underlie the working of the brain.

Practical Classes
These laboratory classes will complement, consolidate and extend what is
covered in the lecture series. You should not however expect a precise match
between lectures and labs because different aspect of Neurobiology are best
suited to different modes of teaching.
Practical classes provide an excellent opportunity to interact more closely
with members of the academic staff and senior students serving as
demonstrators. Attendance of practical classes is essential for this paper and
constitute a terms requirement. Should you miss, without valid reason, three
laboratory sessions you will fail to achieve the terms requirement. Failure to
achieve the terms requirement means you will not be able to sit the final
exam.
IMPORTANT NOTE: Please refer to the relevant Appendix in your
Laboratory Manual, for information on the conditional use of selfdeclaration forms from Student Health, as human cadaveric material will be
used for various components of this paper.

Textbooks
A new textbook has been selected as essential for this paper: Neuroscience:
Exploring the Brain (3rd Edition) by Mark F. Bear, Barry W. Connors and
Michael A. Paradiso (Published 2007 by Lippincott Williams and Wilkin).
This book will be available at the University Bookshop, please enquire
during semester 1 to make sure you get a copy. You should also note that as
a new textbook second hand copies are likely to be hard to obtain. A limited
number of copies of this textbook are available from the Close Reserve in
the Medical library but you will be at a disadvantage if you do not have

ready access to a copy.

At various stages of the paper you will be asked, and indeed expected, to
have read and understood specific sections of this textbook before the
lecture. The following textbooks are available from the Close Reserve in the
Medical library:
Marieb: Human Anatomy and Physiology (7th ed. - 8th ed. is currently on
order and will also be put on Close Reserve).
Eizenberg: General Anatomy: Principles & Applications, McGraw-Hill 2007
Saladin: Anatomy and Physiology: the unity of form and function (3rd Ed.)
A package called Neuroanatomy II has been designed to accompany the
neuroanatomy labs. This package consists of five books: Neuroanatomy II
Book 1 to Book 5. Diagrams contained in them are numbered, e.g. Fig 1; Fig
2 etc. The Neuroanatomy Books are also available via Blackboard.
Assessment
ANAT 242 involves both internal and external assessment. As with our
other 200-level papers 70% of the final mark will be derived from a final 3
hour exam. The remaining 30% will be based on a number of items of
internal assessment (see below). Note again from above, you must meet the
terms requirement for practical classes or you will not be eligible to sit the
final exam.
Assessment 1 (10%): Internal Test 1
Assessment 2 (10%): Internal Test 2
Assessment 3 (10%): Laboratory Assessment

ANAT 242

Objective
- to learn the structure and function of the
nervous system
Anatomical structure & function
Cellular organization

Dr. Ping Liu

Dr. Ruth Napper

Social & ethical ramications

Prof. Gareth Jones

Molecular events
Dr. Ping Liu

Lectures
Mondays & Tuesdays: 11 - 11.50 am
Wednesdays (alternate): 10 10.50 am

Labs (Histology Classroom)

Stream A: Mondays 2 - 3.50 pm
Stream B: Mondays 4 - 5.50 pm
Stream C: Mondays 6 7.50 pm
Compulsory!!!
Today: Museum Introductory Session
(normal stream times)

Assessment Components
Internal assessment (30%)
Test 1 (10%): Week 5
Test 2 (10%): Week 9
Test 3 (10%): week 12
Final examination (70%)

Application for impairment

Terms requirements
(see lab manual for details)

Neuroanatomy

Introduction to brain and spinal cord

Meninges & ventricular system
CNS development & blood supply
Cerebrum
Basal nuclei & Diencephalon
Brain stem & Cranial nerves ( 2)
Cerebellum
Motor systems
10 lectures
Sensory systems
6 labs

Lecture 1: Introduction to the

brain & spinal cord
Objectives
Be able to identify the external features of the brain
Be able to identify/describe the major features of the
spinal cord
Understand the consequences of spinal cord damage

 The CNS is the

integrating and
command center

The PNS is the

communication
system linking all
parts of the body to
the CNS
Saladin Fig. 12.1 (P. 444)

Organization of Nervous System

Central Nervous System
(CNS)

Peripheral Nervous System

(PNS)

(Brain & Spinal Cord)

(Cranial & Spinal Nerves)

Sensory (Afferent) Division

Motor (Efferent) Division

(Somatic & Visceral Nerve Fibers)

(Motor Nerve Fibers)

Autonomic Nervous System (ANS)

Somatic Nervous System

Involuntary (Visceral Motor)

Voluntary (Somatic Motor)

Major subdivisions of the CNS

(Cerebrum)
Brain:

Cerebral Hemispheres
Diencephalon
Midbrain
Pons
Medulla

Hindbrain

Spinal cord

CNS subdivisions
Brain weighs
approx 1.5 kg

Cerebrum is
83% of brain
volume

Marieb, Fig. 12.3

(P. 432)

contains
50% of
neurons
Spinal
cord

Orientation: cerebrum
Superior
(dorsal)
Posterior
(caudal)

Anterior
(rostral)

inferior
(ventral)
Lateral view (left)

Marieb, Fig. 12.3

(P. 432)

Orientation: brainstem & spinal cord

Superior
(rostral)
Posterior
(dorsal)
Anterior
(ventral)

Inferior
(caudal)
Lateral view (left)

Marieb, Fig. 12.3

(P. 432)

Sections

Horizontal

coronal
Lateral view (left)

Sections

Midsagittal
(midline)

From Anatomy Museum

Sagittal

Marieb, Fig. 12.3 (P. 432)

Nerve tissue contains two basic cell types

Neurons (nerve cells)
- highly specialized, excitable cells
- have high metabolic rate
- provide rapid and specic communication
between regions of the body

Neuron - Functional Classication

afferent
incoming

efferent
outgoing
Saladin Fig. 12.3 (P. 446)

Sensing

Thinking

Controlling

Grey & white matter

cortex

Grey matter
(Neuron cell bodies)
Nucleus (CNS)
Ganglion (PNS)

White matter
(Axons)
Lipid material in
myelin sheaths gives
white appearance
From Anatomy Museum

External features of the brain

Dura mater

From Anatomy Museum

External features of the brain

Gyri = hill tops
(gy = turn, twist)
singular form - gyrus
Sulci

Sulci = valleys
(sulc = groove)
singular form - sulcus
Fissure separates large
regions of the brain

From Anatomy Museum

Marieb, Fig. 12.6 (P. 435)

 is buried deep within the

lateral sulcus & forms part
of its oor
is covered by portions of
the temporal, parietal &
frontal lobes
From Anatomy Museum

Superior surface
Frontal Lobe

Central
sulcus
Parietal Lobe

Occipital Lobe
From Anatomy Museum

 Lateral surface

Central
Sulcus

Transverse
ssure
(separates the
cerebrum
from the
cerebellum)

Temporal
Lobe

Lateral
sulcus
From Anatomy Museum

Medial surface
Parieto-occipital
sulcus

Parietal
Lobe

Occipital Lobe
Transverse
ssure
Cerebellum

Diencephalon
Brain stem
From Anatomy Museum

 Ventral surface
Frontal Lobe

Temporal Lobe

Cranial
nerves
+
blood
vessels

Cerebellum
From Anatomy Museum

Spinal Cord Anatomy

Extends from the foramen
magnum to rst or second
lumbar vertebra

(8)

(12)

About the width of your

thumb and ~ 42 cm long
31 pairs of spinal nerves
Provides two-way street
of information to and
from the brain

(5)
(5)
Coccygeal (1)
Marieb Fig. 12.28 (P. 470)

 Two enlargements
- where the nerves serving
the upper & lower limbs
arise

Conus medullaris
(Medullary cone)

- extends from the conus

medullaris to the posterior
surface of the coccyx

Marieb Fig. 12.28 (P. 470)

- the collection of nerve

roots at the inferior end of
the vertebral canal

Why?
After birth the vertebral
column grows faster than
spinal cord
Marieb Fig. 12.28 (P. 470)

Cross-section of spinal cord

Posterior
median
sulcus

Posterior
(Dorsal)
Anterior
(Ventral)
Anterior
median
ssure

Grey matter centrally located

White matter peripherally located

Source unknown

Spinal Cord - Grey Matter

Posterior (dorsal) horns

Anterior (ventral) horns

Source unknown

(interneurons)

(motor neurons
+ interneurons)

Spinal Cord - White Matter

Anterior (Ventral) Columns

Source unknown

sensory impulses
to the cord
Dorsal root
ganglion

Spinal nerve
(mixed)

Source unknown

Ventral root
(efferent bers)

axons to effector
organs

Cervical

Thoracic

Lumbar

Enlarged grey matter in cervical (C3-T1)

and lumbar (L1-S2) regions

Source unknown

Cervical

Thoracic

Lumbar

More white matter in dorsal columns

from lumbar to cervical cord
More sensory bers from the upper limbs added
Source unknown

Spinal Cord Injury (SCI)

Paralysis - loss of motor function
Paresthesias - sensory loss

~260 new spinal cord injuries each year in NZ

80% SCI are males aged 15-35 years

Source unknown

The severity of spinal cord

injury depends on what
level the injury occurs at.
C1-C4 = high tetraplegia
C5-C8 = low tetraplegia
Thoracic, lumbar or sacral
injuries = paraplegia
Injuries can either be complete
or incomplete
Marieb Fig. 12.28 (P. 470)

Q: Spinal Cord Damage

Source unknown

References
Marieb: Human Anatomy & Physiology (6th Ed.)
- Chapters 11 & 12
Saladin: Anatomy and physiology: the unity
of form and function (3rd Ed.)
- Chapters 13 & 14
Bear, Connors & Paradiso: Neuroscience:
exploring the brain.
- Chapter 7

ANAT 242
Neuroanatomy
Lecture 3: CNS Development
& Blood Supply
Dr. Ping Liu

Objectives
Be able to describe the development of CNS
Be able to describe the blood supply and
venous drainage of the brain
Be able to identify the Circle of Willis and
venous sinuses
References
Marieb: Human Anatomy & Physiology
- Chapters 12
Saladin: Anatomy and physiology: the unity of form and function
- Chapter 14
Bear et al: Neuroscience: exploring the brain
- Chapter 7

CNS
Development
11 days

The entire nervous system arises

from embryonic ectoderm
The ectoderm thickens to form
the neural plate
The neural plate folds inward to
form the neural tube
Neural crest gives rise to neurons
destined to reside in ganglia

Marieb, Fig. 12.1 (P. 431)

Epithelial cells lining the neural tube

generate all the neurons of the CNS

Neural tube
the rostral
portion forms
the brain
the caudal
portion forms
the spinal cord
the cavity forms
the ventricular
system

Embryonic Development of Human Brain

Week 5

(endbrain)
(interbrain)

(afterbrain)
(spinal brain)

Marieb, Fig. 12.2 (P. 432)

Brain Development

5 weeks

13 weeks

birth

By birth, cephalic exure (midbrain level) remains prominent

Convolutions - increase the surface area of the brain.
Marieb, Fig. 12.3 (P. 432)

Spinal Cord Development

Marieb, Fig. 12.27 (P. 469)

CNS Development
Although the external shape of the brain &
spinal cord is well developed at birth
after birth

size
synapses

prefrontal cortex development

Early Childhood Education

Neural Tube Defects

Anencephaly
(without brain)
The hemispheres or the

whole forebrain are usually

absent
Almost always accompanied

by severe cleft spinal cord

Because the neural folds

fail to fuse rostrally

The infants are usually born

dead or die soon after birth

Neural Tube Defects

Spina Bifida
(forked spine)
Results from incomplete formation

of the vertebral arches and typically

involves the lumbosacral region
Because the caudal portion of the
neural tube fails to close
Often accompanied by a posterior

midline protrusion including some

of the content of the vertebral canal
Neural problems - depend on the

degrees of malformation

Blood supply of the brain

Brain constitutes only 2% of body weight, but
receives 15% of blood ow (about 750 ml/min)
Brain consumes 20% of bodys oxygen and
glucose
10 second interruption of blood ow can cause
unconsciousness
1-2 minute interruption can cause impaired
neural function
4 minutes interruption causes irreversible brain
damage

Cerebral
Arteries

Basilar
Artery

Marieb Fig. 19.20 (P. 749)

Cerebral Arteries

The basilar artery divides into two

posterior cerebral arteries

Cerebral Arteries
Anterior Cerebral Artery
Middle Cerebral Artery

Internal Carotid Artery

The internal carotid artery branches into

anterior & middle cerebral arteries

Circle of Willis
Basilar artery
connects to internal
carotid artery by
communicating
arteries forming the
Circle of Willis
To equalize blood
flow to various parts
of the brain (?)

Moore, Fig. 7-54

Only 20% people

have complete circle!

Posterior
Cerebral
Artery

Carpenter, Fig. 14.5 (P. 442)

Middle
Cerebral
Artery

Carpenter, Fig. 14.6 (P. 442)

Anterior
Cerebral
Artery

Carpenter, Fig. 14.5 (P. 442)

Stroke
refers to the neurological dysfunction that results
from a reduction of blood supply to the brain
includes both blockade of a cerebral artery (or vein)
leading to cerebral infarction, and
haemorrhage from an artery (or vein)
usually caused by an occlusion of a vessel by blood
clot or cholesterol deposit or bleeding from a
ruptured vessel
The neurological signs and syndromes associated
with a stroke depend on which blood vessels
and their branches are involved.

Occlusion of

Why?

Anterior cerebral artery

- contralateral hemiplegia (one artery) or bilateral
paralysis (two arteries) & impaired sensation
greatest in the lower limb
Middle cerebral artery
- a severe contralateral hemiplegia & impaired
sensation
most marked in the upper limb & face
- severe aphasia (if the dominant hemisphere is affected)
Posterior cerebral artery
- contralateral homonymous hemianopsia

Blood drainage of the brain

ne veins (brain)
pial venous plexuses
cerebral veins
veins (scalp)

dural venous sinuses

Emissary
veins

internal jugular vein

heart

Venous Sinuses

between two layers of dura matter

receive venous blood from brain and scalp
receive CSF
Marieb, Fig. 12.23 (P.463)

Draining the superior & deep structures

1) Superior sagittal sinus
- lies along the superior
margin of the falx cerebri
- joins the transverse
sinus (right )
- Arachnoid villi drain the
CSF into the superior
sagittal sinus

Marieb, Fig. 19.25 (P.761)

Draining the superior & deep structures

2) Inferior sagittal sinus
- lies along the inferior
margin of the falx cerebri
- joins straight sinus

Marieb, Fig. 19.25 (P.761)

Draining the superior & deep structures

3) Straight sinus
- within tentorium cerebelli

Marieb, Fig. 19.25 (P.761)

- joins left transverse sinus

Draining the superior & deep structures

4) Transverse sinus

- left with straight sinus

Marieb, Fig. 19.25 (P.761)

Question 1
i) Name the structure
labelled A
B

ii) Name the structure

labelled B
iii) What function is B
normally associated with?
iv) Name the structure
labelled C
v) Name the structure
labelled D

A
C
D

Question 1 - answers
i) Name the region
labelled A...
vermis

ii) Name the structure

labelled B...
dentate nucleus
iii) What function is B
normally associated with?...
motor - planning,
initiation, control
iv) Name the structure
labelled C...
middle cerebellar peduncle
v) Name the structure
labelled D...
flocculus

A
C
D

Question 2
i) Name the structure
labelled A
ii) Name the region
labelled B
iii) Name the region
labelled C

A
D

iv) Name the structure

labelled D
v) Name the structure
labelled E

Question 2 - answers
i) Name the structure
labelled A...
inferior colliculus
ii) Name the region
labelled B...
pons
iii) Name the region
labelled C...
cerebellar cortex/
hemisphere

A
D

iv) Name the structure

labelled D...
cerebral aqueduct
v) Name the structure
labelled E...
inferior medullary velum

Question 3
i) Name the tissue type
shown in this image

B
C

ii) Name the tissue

labelled A (surrounding
the small circular
profiles)
iii) Name the tissue
labelled B
iv) Name the tissue
labelled C
v) What glial cells could you expect to find in this tissue type?

Question 3 - answers
i) Name the tissue type
shown in this image...
peripheral nerve

ii) Name the tissue

labelled A (surrounding
the small circular
profiles)...
endoneurium
iii) Name the tissue
labelled B...
perineurium
iv) Name the tissue
labelled C...
epineurium
v) What glial cells could you expect to find in this tissue type?...
Schwann cells

Question 4
i) Name the structure
labelled A

ii) Name the structure

labelled B
iii) Name the structure
labelled C
iv) What cell type
produces the profile
labeled C?
v) Name the overall
tissue type shown in this image

Question 4 - answers
i) Name the structure
labelled A...
Schwann cell cytoplasm

ii) Name the structure

labelled B...
axon
iii) Name the structure
labelled C...
myelin
iv) What cell type
produces the profile
labeled C?...
Schwann cell
v) Name the overall
tissue type shown in this image...
peripheral nerve

Question 5
i) Name the structure
labelled A

ii) Name the structure

labelled B
iii) What would be
occupying the spaces
marked with asterisks?
iv) What are the small
circles in the profile labelled C?

*
D
B

v) Is the profile labelled D pre- or post-synaptic?

Question 5 - answers
i) Name the structure
labelled A...
dendritic spine

ii) Name the structure

labelled B...
mitochondrion
iii) What would be
occupying the spaces
marked with asterisks?...
glial cells, processes
of other neurons

iv) What are the small

circles in the profile labelled C?...
synaptic vesicles
v) Is the profile labelled D pre- or post-synaptic?...
post-synaptic

*
D
B

LAB 5

NEUROANATOMY 5
CEREBELLUM, CELLULAR STRUCTURE OF CNS TISSUE and
PERIPHERAL NERVES

1. CEREBELLUM (Neuroanatomy II Book 3 Figs 7-9)

The cerebellum, like the cerebrum, has two hemispheres, left and right. The
hemispheres are connected medially by the vermis. The surface of the cerebellar
hemispheres is folded, the ridges formed by these folds are known as folia (leaves).
Each hemisphere has three divisions; anterior and posterior lobes divided from each
other by the primary fissure and the flocculonodular lobe, which is made up of the
flocculi (found in each hemisphere) and the nodule (found in the midline at the end of
the vermis). (Figure NA 5.1). Functionally the anterior and posterior lobes are
important in movement coordination and the flocculonodular lobe is important for
adjustments of posture to maintain balance.
With which lobes of the cerebral hemispheres is the cerebellum in contact?
Basal portions of the occipital and temporal lobes.
Which brainstem regions are overlapped by the cerebellum?
Pons and medulla.

The ventral aspect of the cerebellum rests on the foramen magnum; a shallow circular
groove on the cerebellar surface indicates that relationship. The portion of the
cerebellar cortex between that groove and the medulla is the tonsil. In pathological
conditions with rapidly increasing intracranial pressure, the tonsils may be pushed
between the medulla and the edges of the foramen magnum. Death can occur by
compression of the centres of the medulla controlling respiration and blood pressure.
A horizontal section of the cerebellum, shows the arrangement of the white matter as
the arbor vitae (See: Fig. NA 5.2; Neuroanatomy II Book 3, Fig. 9). Note that the
white matter is covered by a thin layer of cerebellar cortex (gray matter) which covers
all the surface of the cerebellum.
There are four nuclei embedded within the white matter of the cerebellum (Figure NA
5.2). These nuclei are known as the fastigial, emboliform, globose and dentate nuclei.

83

LAB 5

Only one of these nuclei - the dentate nucleus is large enough to be seen with the
naked eye. Identify the dentate nucleus in model NSC2.19 or a coronal brain section.
The other nuclei are seen only in histological preparations.

Figure NA 5.1: Gross external structure of the cerebellum. A. superior view and B. Inferior
view.

Figure NA 5.2: Schematic showing the location of the deep cerebellar nuclei.

84

LAB 5

The cerebellum is attached to the brainstem by the three cerebellar peduncles

(Neuroanatomy II Book 3 Fig. 7).

Locate these peduncles but note that only the middle peduncle is clearly visible in
the intact brain. The inferior and superior peduncles are more readily seen in isolated
cerebella.

Middle cerebellar peduncle, which is continuous with the pons.

Inferior cerebellar peduncle, by which the cerebellum is attached to the

dorsolateral aspect of the medulla.

Superior cerebellar peduncle, arising from the dorsal surface of the midbrain,
under cover of the occipital lobes.

Also locate:

Superior medullary velum (Figure NA 5.6), which is more substantial than the
inferior, and is visualised by elevating and retracting the superior part of the
cerebellum; it roofs in the space between the converging superior cerebellar
peduncles and the cerebellum.

Inferior medullary velum, or its remnants, roofing in the gap between the
diverging inferior peduncles and the cerebellum. Note this is not always
visible.

Fourth ventricle, expanding on the dorsum of the brainstem under cover of the
cerebellum (Neuroanatomy II Book 3 Fig. 1). This ventricle can only be seen
in specimens with the cerebellum removed.

How are the superior and inferior medullary vela related to the fourth ventricle?
These structures form the roof of the fourth ventricle.

85

LAB 5

Figure NA 5.6: Midline view of the brainstem and cerebellum.

2. THE CELLULAR COMPOSITION OF THE CEREBELLUM

From a Demonstrator or technician, obtain a slide of monkey cerebellum stained
with thionin, which is a Nissl stain (GB122). View the section by eye and then
under the light microscope. (When you have finished using the slide, please
return it to the central table.)
Identify the complex tree like structure, the arbor vitae, that the white matter forms as
the internal structure of the cerebellum and the layer of gray matter that forms the
outer covering of the cerebellum.
Under the microscope:
View the gray matter and identify a range of neurons, note that these have a variable
form and from distinct layers.
What structures will occupy the space between the neuronal cell bodies?
Other cells such as glia, other processes such as dendrites and axons.
View the white matter and identify nuclei in the white matter that lies immediately
beneath the gray matter.
What cell type do these nuclei belong to and what is their function?
Oligodendrocytes - myelinate axons of the CNS.

86

LAB 5

The transmission electron micrographs below (TEM 5.1 and 5.2) show two images
taken from the gray matter of the cerebellar cortex; they do not include any cell
bodies.

TEM 5.1: A Purkinje cell spiny dendrite from the cerebellar cortex of the rat. (magnification
= x 16,867)

TEM 5.2: A Purkinje cell spiny dendrite from the cerebellar cortex of the rat. (magnification
=x 27,125)

One thing that is difficult to comprehend when looking at images of CNS structures
taken in the transmission electron microscope is the real size of them. To help you
understand the real size of structures we will measure some profiles and calculate
their real size.
87

LAB 5

Identify the spines that are attached to the dendrites in TEM 3.1 and 3.2 and number
the spines. Measure the length of each spine.
NOTE: Remember that the two micrographs have different magnifications.
Length of dendritic spines.
spine #

measured length (mm)

actual length (mm)

actual length (microns)

Range of spine length:

Give 2 reasons (at least) that explain why the spines are of different lengths?

Different contacts being made by the spines.

Different planes of section.

3. THE CELLULAR STRUCTURE OF CNS TISSUE

GENERAL OBJECTIVE:
To be able to recognize the key features of nervous tissue in light and electron
microscopic images and to relate these features to the function of the nervous system

3.1 Cellular Components of the CNS.

Examine Slide 9 (Neuro box) - Sensory Motor Cortex of Baboon, Nissl stain.
This contains a section through a part of the cerebral cortex, the sensory motor cortex
of the baboon.

88

LAB 5

Use this section to show you the layered arrangement of cells in the outer gray matter
of the cerebrum and note that it forms a highly folded later over a core of white
matter. Identify the gray matter and the white matter.
What type of fibres would be found in the white matter underlying the gray matter?
Myelinated/unmyelinated axons local cortico-cortico projection fibres.
There are two predominate types of nucleus in this white matter.
What cell types do they belong to?
Oligodendrocytes, endoflelial cells (blood vessels), astrocytes.
View the gray matter with the x4 objective lens and note that it is arranged in layers
and between each layer the density of neurons and their size changes.
Identify examples of neurons, astrocytes and the endothelial cells of capillaries.
List the features that you have used to establish the identity of each cell type.
Make sure you can actually see these features rather than knowing that is what you
should see.
Neuron stained cytoplasm (processes; nucleus in soma large/pale).
Astrocyte nucleus only (speckled).
Blood vessels flattened endoflelial nuclei.

The most common way to get an overview of the appearance of the cellular structure
of the brain is to use the Golgi method of staining. This method for staining nervous
tissue was discovered by the Italian physician/scientist Camillo Golgi (1843-1926)
and has been used from that time to study the arrangement of the processes of
neuronal cells in different regions of the brain. The Golgi stain selects cells at random
and impregnates and stains the entire cell such that the entire dendritic tree and all
axonal ramifications can be followed. Commonly the axon will leave the section
containing the cell body. (NOTE: It is more common for neurons to be studied but
glial cells are also stained.) (Sometimes parts of the vasculature are also stained, they
have the appearance of solid black stained smooth processes.) The Golgi method is
particularly useful for determining the arrangement of dendritic and axonal processes,
on individual neurons, and how this changes during life (e.g with adolescent
development, drug treatments, learning, environmental enrichment).

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A human neocortical pyramidal

neuron stained via Golgi
technique. Notice the apical
dendrite extending vertically
above the soma and the numerous
basal dendrites radiating laterally
from the base of the cell body.
The arrow indicates the axon
leaving the cell body, note the
difference in appearance of the
axon to the dendrites.

Collect a slide, GB137, from the special class set on the front bench. This slide
contains a 200 micron thick section of Golgi stained tissue from the right hemisphere
of a 90 day old rat.
Under the x4 objective lens orientate the section so that you can identify the midline
of the forebrain, the curved outer surface of the hemisphere with cerebral cortex, the
outermost layer of the cerebral cortex, and inner mid brain structures. Note that each
of you will have a different section, some will be very rostral while others will be at
around the mid point of the rostral caudal axis of the forebrain.
Note that the different regions of the brain show a different pattern of staining.
Using the x10 objective lens identify the cerebral cortex, that in most cases, except
the most rostral sections, is underlain with the white matter of the corpus callosum.
Look at the layered arrangement of cells in the cerebral cortex and describe what you
can see below.
Describe the arrangement of the layered cerebral cortex and note how it differs
from the layering you looked at in the Nissl stained, gray matter, of slide 9.
A distinct layer of labelled cells in the more superficial part of the cortex. The cell
soma are small compared to cells that are located more deeply. The staining seems
more sparse in the deeper layers but that is due to the cells being larger. Can still
see different patterns of staining as you go from the medial to the lateral portions
of the cerebral cortex indicating that the shape and arrangement of the cells are
changing. This is representative of the changes in cell density and cell soma size
that is seen in a Nissl stained section.

Using the x20 objective lens view the cells in the outer layers of the cerebral cortex.
Find a neuron that has a number of dendrites and an axon leaving the cell body. You
will need to focus up and down through the section to identify the continuity of
dendritc and axonal processes within the thickness of the section.

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List the features that you can see in the Golgi stained preparation that were not visible
in the Nissl stained tissue of slide 9.
Axon and dendrites, also spines on the dendrites.
Only see a few of the cells but see the processes leaving the cell body.

Why were you not able to see these structures in the Nissl stained section?
The dendrites are not visible because they only have a few organelles and in
particular a lack of endoplasmic reticulum, the main element stained in the cell
soma with the Nissl stain. If there is nothing for the stain to bind to there will
be no staining. The axons have even fewer organelles, cytoskeleton and a few
mitochondria so would not be visible in bright field light microscope with a
Nissl stain.
Using the x40 objective lens focus on a denritic branch. Choose a branch that is the
second branch after the dendrite has left the cell soma. Focus up and down on this
branch and make sure you can see the dendritic spines.
List the features of the dendritic spines you could measure/record in this Golgi
stained tissue?
The number of spines, the shape and size of the spines.
Using the x10 objective lens, move around the section to find a portion of the blood
vascular system that has been stained with the Golgi solution.
It will appear something like this.

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AT SOME TIME DURING THIS LABORATORY CLASS carry out the task
below:
As we have only one microscope set up for this, you will need to have turns.
Go the front bench and use the light microscope that has the eyepiece digital camera
attached. There is a Golgi stained section, similar to the one you have to view under
the microscope and in this task you are required to find structures in the slide and
print out a photograph of them.
Below are examples of the structures that you need to find, capture an image of and
print this image out.

Image 1
Image 2
1: Using the x20 objective lens, in the outer layers of the cerebral cortex find an
example of a Golgi impregnated neuron with several dendrites clearly visible in a
single focal plane. Take an image of this cell and print it out.
2: Using the x40 objective lens, in the outer layers of the cerebral cortex find an
example of a Golgi impregnated dendrite with numerous spines that are visible in a
single focal plane. Take an image of this cell and print it out. Return to your bench
with your images and work on the images.

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On your image from 1, label

The soma, the primary dendrite and a secondary dendrite.
List all the structures that you may find within an area indicated by the circle in
Image 1.
There will be more neurons but these are ones that have not been stained with the
Golgi stain. The stain impregnates only a small percentage of the cells and we have
no idea how cells are selected to be stained. It is not known why one cell is stained
and a neighbouring cell is not. It has been shown however if the impregnation time is
sufficient the cell will be stained in its entirety, all its spines to the tip of the spine. It
does appear that staining begins at the soma and moves out along the processes.
Where impregnation has halted part way along a process it appears as a rather sharp
line, however a sharp end to the staining may also mean the process has left the
section, focus to check, or that the process has severed, often you can see a break and
the process then continues, this is most likely to have occurred during sectioning.
There will also be glial cells and elements of the blood vascular system in this
unstained space.
On your image from 2, label
The dendritic branch, label the spines.
What features of the spines are visible in your image.
Can see a variety of shapes and lengths

Are you seeing all the spines on this length of dendrite and if not, why not?
You will not see spines that are behind the branch or are standing out at right
angles to the surface of the branch. Remember that some of the spines that appear to
be short may be coming from the mid point of the branch and extending out laterally
until they are seen.
Make sure a demonstrator has checked your images.
In viewing Slide 9 in the light microscope you will have noticed that only a small
proportion of the gray matter is occupied by the soma of neuronal and glial cells. The
tissue not occupied by cell bodies is often referred to as the neuropile.
The next component of this class uses images taken in the transmission electron
microscope to investigate the structure of the neuropile.

3.2 Dendrites in the neuropile of the central nervous system.

Examine the TEM image on the next page which shows an area of neuropile from the
cerebellar cortex of a rat.
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TEM 5.3: Neuropile from rat cerebellar cortex (x27,403).

The profile labelled D is a large dendrite belonging to a Purkinje cell.

Assuming that this profile shows the widest part of the dendrite, measure the width of
the dendrite what is the real diameter of the dendrite?
Measured diameter (in mm)
Real diameter (in mm)
Real diameter (in microns)

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Identify and label the following structures on TEM 5.3:

Mitochondria - these tend to be long and slender with their long axis running along
the long axis of the dendrite.
Profiles of endoplasmic reticulum - these tubules tend to run longitudinally along
the dendrite and branch to form a system of tubules.
Microtubules and neurofilaments - also oriented along the long axis of the dendrite.
An axon is adjacent to the left side of dendrite.
What identifies this structure as an axon?
Mitochondria (and vesicles).
Identify where the axon is forming a synaptic junction on the dendrite.
What structures, visible in the micrograph, indicate the synapse?
Post-synaptic densities, vesicles, presynaptic densities.

Note that synaptic vesicles are only found close to the synaptic specialization.
How are the vesicles held at this location within the axon?
Via filament and microtubule networks:
- mainly actin filaments to vesicle
- form on actin filament network to plasma membrane.

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3.3 Synapses in the neuropile of the central nervous system.

Examine the TEM image (below), which shows an axonal swelling (known as a
varicosity or bouton) filled with vesicles. This axonal swelling has 3 synapses
formed on it by 3 different dendritic spines. This example is from the gray matter of
the cerebellar cortex.

TEM 3.3: Axonal swelling showing vesicles (x56,224).

Select a synapse and identify and label the following structures that are characteristic
of the synapse:

96

The presynaptic axonal portion.

The postsynaptic portion.
Mitochondria in the axonal portion.
Synaptic vesicles in the axonal portion.
The parallel pre- and post-synaptic areas of membrane specialization and the
synaptic cleft.

LAB 5

Would the protein structure of the pre and post synaptic membrane be same?
Explain what general functional classes of protein you would expect to find in each
of these locations.
No:
Presynaptic:
- re-uptake transporters
- docking proteins
- mind the gap protein binds post synaptically after being secreted across the
matrix presynaptically
Post synaptic:
- receptor proteins / ion cannels
- cell adhesion molecules (adhere MTG protein)

Name the structure indicated by the arrow?

Glial cell (astrocyte cytoplasm).

Name two important functions of this structure at this location.

- re-uptake of transmitter (not used)
- bind transmitter (detect level of activity at synapse)
- release glutamine (recycled glutmate)

Within a myelinated axon identify:

a mitochondrion
microtubules
neurofilaments
the myelin sheath
the cytoplasm of the myelinating glial cell

Identify a number of unmyelinated axons. Note that in some places the

unmyelinated axons are lying directly against each other but in some other regions are
separated, the material separating them is glial cell cytoplasm.
What type of glial cell will this be?
Astrocyte.

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LAB 5

List some of its possible functions of the glial cell at this specific location.
Mopping up excess transmitter.
Provide energy for neuron.
Contains receptors to detect the level of activity of the neuron.
Maintain extracellular potassium concentrations.
Within an unmyelinated axon identify:

mitochondria
microtubules

Why does the cytoplasm of the axons have microtubules?

Transport, neuro filaments for strength.
Explain one method that could be used to definitively identify microtubules in a
transmission electron micrograph.
Immunogold labelling of the microtubule network associated proteins antibodies to:
- MAP 3 / tau (in axon)
- MAP 2 (in dendrites / soma)
Measure the diameter of the smallest unmyelinated axon in the electron
micrograph.
What is the real diameter of this unmyelinated axon?
diameter of unmyelinated axon in micrograph(mm) =
real diameter of unmyelinated axon = in mm
real diameter of unmyelinated axon = in microns

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How does an unmyelinated axon in the CNS differ from one in the PNS? (See
TEM 5.4 below for an example of tissue from a peripheral nerve.)
CNS unmyelinated axon is not covered / surrounded by glial cell processes.

TEM 5.4: Shows an area of a peripheral nerve.

On TEM 5.4, label myelinated axons, an unmyelinated axon, Schwann cell
cytoplasm, endoneurium and FCT of epineurium.
A nucleus is labelled with the arrow, what type of cell does this belong to?
Schwann cell.
A TEM montage of neural tissue can be seen on BLACKBOARD in your study
time - it is called Brain tissue - Zoomify.
The image is of tissue from the gray matter of the cerebellar cortex of the rat.
The image is a montage assembled from multiple overlapping images taken
using the transmission electron micrograph. The single montage has been
arranged so that you can move around it, increasing and decreasing the

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LAB 5

magnification in a way very similar to using the transmission electron

microscope in real time.
Use this resource to explore the structure of the gray matter and find examples
of cellular structures discussed in the laboratory class and lectures.

4. PERIPHERAL NERVES
4.1 The generalized structure of a peripheral nerve.
To examine the structure of a peripheral nerve we will look at sections from the
human plantar digital nerve.
This is quick recap on some work that you did in ANAT 241. It is important to
revise this as you are now thinking of the nerve fibres as an entity doing a
functional task within the CNS.
Examine Slide 44 (Neuro box) - TS & LS human plantar digital nerve.
This contains two sections through the nerve, one where the nerve has been cut in
cross section and one where it has been cut in longitudinal section. This section has
been stained with osmic acid to stain the myelin sheaths black. The connective tissue
has stained a greenish brown.
Using the x4 objective lens study the section where the nerves are cut in transverse
section.
The nerve fibres, i.e. the axons and their associated non neuronal cells, run in bundles
that are enclosed by support tissue that forms a distinct sheath. A number of bundles
lie together to form an anatomical nerve.
Draw a diagram of the section as you see it down the microscope and label
components as described below.

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LAB 5

Identify the epineurium; this forms the support tissue (FCT) between the individual
fasciculi (nerve bundles). This may also form an external sheath around all the
bundles of nerve fibres that make up the anatomical nerve.
With the x10 objective lens note that each fascicle has its own surrounding sheath, the
perineurium. Identify blood vessels between and within the fasciculi.
With the x40 objective lens look at the contents of a fasciculus (bundle) and find an
area where the axons have been cut transversely, i.e. you can see circular profiles of
black stained myelin sheaths. Note that the thickness of the myelin sheaths varies
between individual fibres. The almost clear space within the sheath is the axon.
The lightly stained tissue between the myelinated axons is composed of:

support tissue (FCT) forming the endoneurium,

the cytoplasm of the Schwann cells,
unmyelinated nerve fibres.

Using the box draw a diagram labelling these structures.

Look at the section of the nerve cut in longitudinal section. Try and identify 2 nodes
of Ranvier and the intervening nodal distance on a single nerve fibre.
Describe the significance of the node of Ranvier for transmission of the action
potential along a nerve fibre.
Exposed areas of the axolemma which allow for efficient saltatory conduction.
What are the consequences of demyelination on the transmission of the action
potential?
Decreased efficiency of AP conduction, causing impairment in sensation, movement,
cognition, or other functions depending on which nerves are involved.

NB: SELF-ASSESSMENT QUESTIONS REGARDING THIS LAB WILL BE

AVAILABLE ON BLACKBOARD AFTER TODAYS LAB SESSION.

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Anat 242
Molecular Neurobiology Lab. (2009)
Notes for Demonstrators
Experiment 1: Immunocytochemistry.
Question 1.

A tissue known to express Hepatin 1 and 2, i.e. the liver.

Question 2.

Secondary antibody only (detects any immunoreactivity not due to primary).

Primary antibody pre-absorbed with immunogen (i.e with hepatin 1 or 2).
Preimmune sera.
A tissue known to be negative for hepatin 1 or 2 immunoreactivity.

Results 1

Immunostaining patterns provided as a separate sheet to be given to students.

Results 2

Students to comment on staining patterns

Hepatin 1 (low mag) individual cells around 3rd ventricle (hypothalamic
paraventricular nucleus extending towards median eminence).
Hepatin 1 (medium mag) individual cells with cytoplasm and processes
stained, note granular appearance of staining (this is a fluorescence staining
whereas other images use a chromogen)
Hepatin 1 (high mag), dark staining shows hepatin 1 immunoreactive
processes contacting other cells. Looks like synaptic contact but need EM to
show this. Note this second cell is labelled with a different antibody
(demonstrates the use of dual-labelling).
Hepatin 2 (low mag) widely distributed cell bodies all over brain region
(cortex, although this is not evident from image)
Hepatin 2 (med mag) highly branched cells with spidery processes. look like
astrocytes but have to be careful identifying on the basis of shape.
Hepatin 2 (high mag) confirms astrocyte form. staining in cytoplasm (not
granular). If believed to be astrocytes how would this be checked? (use an
astrocyte specific stain to dual label).

Additional experiments
1. Hepatin 1 staining seen in granular bodies (about 100 nm dia) located in nerve terminal and
axon.
2. Hepatin 2 staining seen throughout human brain tissue but particularly intense over region of
glioma
3. Hepatin 1 staining is decreased in aged rats whereas hepatin 2 staining shows a possible increase.

Dear ANAT242 students

Some students have asked for further clarification as to the examinable content and the
examples of test questions (as the past exams are embargoed) for the final examination
scheduled for Thursday 4 November.
Components
-30 multiple choice questions (10%)
- Modules 1, 2 & 4
- structured answer questions (15%)
- Module 1 only
- short essays (45%)
- Modules 2, 3 & 4
* Please note that both lectures and laboratory material are examinable in the final
exam.
Examples of MCQ
1) Which of the following statements about xxxxxx is CORRECT (or INCORRECT)?
A-E
2) The structure labelled A on the diagram below is:
A-E
Example of shorter answers
Complete the tables below:
Structure(s)

Function
Location
Components

Examples of essay questions

1) Basal nuclei may function as a break in the motor control. Discuss this statement.
2) Describe the three levels of the motor control. Your answer should include a brief outline
of the structures that are involved in the motor control, and a brief discussion of the roles of
these structures.

Best wishes for the examination!

Regards
Ping Liu
ANAT242 Coordinator