AEB Practical Guide

MFAC1526 Ageing and Endings B
Practical Manual
Session 2: TP4 2010

September 2010

Contents

Preparation and Behaviour in Practical Classes ................................................................................... 3
Practical Schedule ................................................................................................................................ 5
Practical 1: Chest wall, Axilla and Breast ............................................................................................. 6
Practical 2: Analysis of cell ageing ....................................................................................................... 9
Practical 3: Histopathology of Breast Lumps ..................................................................................... 14
Practical 4: Cranial Cavity and Introduction to the Brain .................................................................. 18
Practical 5: Brainstem ........................................................................................................................ 20
Practical 6: Membrane Potentials...................................................................................................... 23
Practical 7: Oestrogen receptor binding ............................................................................................ 31
Practical 8: QMP: Weighing the Evidence .......................................................................................... 36
Practical 9: Cranial Nerves ................................................................................................................. 42
Practical 10: Somato‐sensory System ................................................................................................ 44
Practical 11: CNS: Normal and abnormal ........................................................................................... 50
Practical 12: Internal Capsule and Horizontal Slices of the Forebrain ............................................... 55
Practical 13: Motor Systems Physiology: Reflexes............................................................................. 57
Practical 14: Human aspects of living with neuro‐degenerative disease .......................................... 64
Practical 15: Coronal Slices of the Forebrain ..................................................................................... 65
Practical 16: Visual Physiology ........................................................................................................... 67
Practical 17: CNS Pharmacology ........................................................................................................ 73
Ageing & Endings B
Practical Manual 2010
Page 2

Preparation and Behaviour in Practical Classes

• You are required to familiarise yourselves with the appropriate section of the practical manual before
attending each class.

• In the interests of your safety, special attention should be paid to any precautionary measures
recommended in the notes. If any accidents or incidents occur, they should be reported immediately to
the demonstrator in charge of the class who will record the incident and recommend what further action
is required.

• You must take due care with biological and hazardous material and make sure all equipment is left clean
and functional.

• No eating, drinking or smoking is permitted in the teaching laboratories.

• A white laboratory coat must be worn to those practical classes identified by an asterisk * in the science
practical schedule. Coats must be removed upon leaving the laboratory class. Enclosed shoes must be
worn to ALL classes.

• You are expected to be punctual. Those who arrive more than 10 minutes late may not be admitted to the
class.

• When participating in Anatomy laboratory classes

1. Gloves should always be worn when handling the specimens in the dissecting room. You should
provide your own gloves.

2. Proper care should be taken when handling specimens. The appropriate probes should be used
when demonstrating. Please do not grip delicate tissue with serrated forceps. All gross specimens
should be covered with a damp towel at the end of the class. Each specimen represents an
enormous investment of staff time and money and must be treated with care. Nerves and small
vessels are particularly prone to damage by desiccation.

3. You should place any rubbish into the appropriate waste receptacle. This applies to both the
dissecting room and histology teaching laboratories. If rubbish is left in the histology teaching
laboratories, those labs may be closed to revision.

NB: Those who do not adhere to these basic laboratory rules will be excluded from the class and marked
absent.

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ATTENDANCE REQUIREMENTS

• Given the considerable time, effort and cost involved in the design, preparation and delivery of
practical classes, attendance is expected at all practical classes/demonstrations by all
students.

• A roll will be marked in some classes.

• You are also expected to attend only the class to which you have been rostered. Teachers can and
will exclude from a class any student who is not rostered to attend, unless they have written
approval from the principal teacher to be there.

Requests for changes to Laboratory times for Biochemistry, Genetics, Microbiology and Molecular
Biology Practicals
This notice applies to ALL Phase 1 practical classes held in the School of Biotechnology and
Biomolecular Sciences (BABS) laboratories – Rooms 107 and 107D, Biological Sciences Building.

Please note that there will be NO pre‐arranged changes to times for attending these practical
classes.

Each laboratory can accommodate up to 56 students in 4 groups of 14. Students wishing to change
practical classes should arrive early and consult the member of academic staff in charge at the start of
the practical session they wish to attend. If there are places available in any of the groups, students
with genuine reasons for a change of lab will be allowed to do the practical at that time. If there are
more students wishing to change to that time than there are places available, places will be allocated
on a “first come” basis. The total number of students in each laboratory will not be allowed to exceed
56. Under no circumstances will a student who is attempting to change practical classes be
accommodated if they turn up late to the class they wish to attend.

Please do NOT email the course co‐ordinators or academic staff in BABS about changes to practical
times. Such emails will not be responded to.

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Practical Schedule

This schedule is subject to change. Refer to the eMed Timetable system and email updates sent to your UNSW
email account for times and locations.

Science Practical Principal Teacher
1 Chest wall, Axilla and Breast* Prof. Ken Ashwell
2. Analysis of cell ageing * Dr Louise Lutze‐Mann
3. Histopathology of breast lumps A/Prof Gary Velan
4. Cranial cavity and brain* Dr Elizabeth Tancred
5. Brainstem* Dr Elizabeth Tancred
6. Membrane potentials* Dr Andrew Moorhouse
7. Oestrogen receptor binding* Dr Anne Galea
8. QMP: Weighing the Evidence Dr Rachel Thompson
9. Cranial Nerves* Dr Elizabeth Tancred
10. Somato‐sensory System Dr Richard Vickery
Mr Patrick de Permentier /
11. CNS: Normal and abnormal
Dr Gary Velan
12. Internal Capsule and Horizontal Slices of the Forebrain* Dr Elizabeth Tancred
13. Motor Systems Physiology: Reflexes* Dr Paul Bertrand
14. Human aspects of living with neuro‐degenerative disease Dr Úte Vollmer‐Conna
15. Coronal slices of the forebrain* Dr Elizabeth Tancred
16. Visual Physiology Dr Richard Vickery
17 CNS Pharmacology* Dr Nicole Jones

* Denotes those classes win which laboratory coats must be worn.
Enclosed shoes must be worn to all classes.
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Practical 1: Chest wall, Axilla and Breast
Principal Teacher: Prof. Ken Ashwell
Specific objectives:
In the limited time dedicated for this practical class, you are expected to know only the anatomical features
listed in the following practical guide. Use the materials from the lecture on the chest wall and axilla, and the
lecture on the lymphatic system, and read the recommended reading material to prepare for this practical
class.

Watch the video at the beginning of the class and work your way through the various stations answering
question in the notes below. Please note that you do not need to work through the stations in numerical
order. The answers to the questions may be found in your lecture notes or your textbook. Staff resource
persons will assist you if you are unable to find listed structures on your own. They will ask you questions to
challenge you and stimulate your learning. A mini‐spot test will be available for you to test your knowledge
at the end of the class.

I. THE CHEST WALL
The thoracic or chest wall is formed by the thoracic part of the vertebral column (thoracic spine), the sternum,
ribs and costal cartilages, and the muscles, which fill in the gaps between the ribs (intercostal muscles) and
cover the thoracic cage.

Station Instructions and Notes Questions
1 Review the major features of a typical thoracic vertebra and Which vertebral joints are
identify the following: body, costal demifacets, pedicle and involved in joints between
lamina, articular, transverse and spinous processes. Identify the vertebrae? How do the parts of
articular surface on the transverse processes for the tubercle of the thoracic vertebrae protect
the corresponding rib. the spinal cord and spinal
nerves?
2 Review the major features of a typical rib and identify: head with What type of joint is found
articular facets, neck, angle, shaft, and tubercle with its articular between the manubrium and
and non‐articular components. Use your lecture notes to body of the sternum? What is
consider how the head and tubercle of a rib articulate with the this point called?
body and transverse process of the thoracic vertebra What structures lie within the
(costovertebral and costotransverse joints). costal groove?
Identify parts of the sternum: manubrium, body and xiphoid
process.
3 On the articulated skeleton, observe the general shape of the Which costal cartilage articulates
thoracic cage (truncated cone, elliptical on cross section). Note with the sternal angle?
that the transverse diameter of the thorax increases gradually
from the first to eight or ninth rib.
Note that the cartilages of the ribs 1‐7 articulate directly with the
sternum (true ribs) via costal cartilages, the cartilages of ribs 8‐10
articulate with cartilages immediately above them (false ribs),
and ribs 11‐12 are free (floating ribs). The costal margin is
formed by the 10th rib and the anterior ends of ribs 7‐10. Note
that a typical rib runs anteriorly and downwards.

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4 Identify, by palpation on yourself and your colleagues, the
following reference landmarks that are used in clinical
examination:
• First rib
• Sternal angle
• Xiphoid process
• Costal margin
• Midclavicular line
• Anterior and posterior axillary lines (and folds)
• Midaxillary line
5 On the specimen of the posterior chest wall, identify the three What are the directions of
layers of muscle (the external, internal and innermost muscle fibres in the three layers?
intercostals) in an intercostal space. The external intercostals do
not reach the sternum, and the internal intercostals only extend
posteriorly to the angles of the ribs. Note the direction of the
intercostal muscle fibres. The external oblique fibres run
inferiorly and anteriorly, whereas the internal oblique fibres run
anteriorly and superiorly.
6 Identify the intercostal nerve and vessels (posterior intercostal Between which muscle layers do
artery and vein) that lie in the plane between the internal and the neurovascular elements run?
innermost intercostals (vein, artery and nerve from above
downwards). The intercostal nerves have lateral and anterior
cutaneous branches.
The blood supply to the chest wall is derived from the posterior
intercostal arteries with their lateral branches, and the anterior
intercostal arteries arising from the internal thoracic arteries
(from respective subclavian arteries).

II. HUMERUS and MUSCLES OF THE AXILLARY WALLS AND ARM
Use the bones and wet specimens for the following stations.
7 Use the isolated humerus and articulated skeleton to identify key What structures pass through the
bony landmarks at this bone’s upper end. Note the greater and cervicoaxillary canal?
lesser tubercles, head, anatomical neck and surgical neck and
the intertubercular groove with its medial and lateral lips. On
the articulated skeleton, outline the medial, lateral and posterior
walls of the axilla and the superior entrance into the axilla
(cervicoaxillary canal; bounded by the first rib, clavicle and upper
border of the scapula).
8 Use the wet specimens to identify pectoralis major, pectoralis What are the functions of the
minor, latissimus dorsi, subscapularis, teres major and serratus muscles in bold to the left?
anterior. Use your lecture notes, atlas or the specimens to
determine the attachments of pectoralis major, subscapularis,
teres major and latissimus dorsi on the humerus. On the
prosected specimens, identify the coracobrachialis, the long and
short heads of biceps brachii, and discuss the attachments of the
above with colleagues.
9 On favourable specimens you may be able to see branches of the In what sort of surgery might
brachial plexus to the muscles that bound the axilla (long these nerves be damaged?
thoracic nerve to the serratus anterior, pectoral nerves to the
pectoralis muscles, subscapular nerve to the subscapularis
muscle.

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III. THE LYMPHATIC SYSTEM AND AXILLARY LYMPH NODES
Refer to your lecture notes concerning the general organisation of the lymphatic system.
10 Note that axillary lymph nodes have been removed from the Which lymph node group would
prosected specimens along with loose connective tissue, but you first receive lymph from the arm?
should be able to use your lecture notes to indicate to colleagues Which would first receive lymph
the expected positions of the five groups of axillary lymph nodes from the breast? Why are lymph
(central, apical, lateral or humeral, anterior or pectoral, and nodes enlarged when there is an
posterior or subscapular). Use your lecture notes to recall the infection or cancer in the area
pattern of lymph drainage between these groups. that they drain? How does
palpation of enlarged lymph
nodes help differentiate between
infection and cancer?

IV. THE MAMMARY GLAND (THE BREAST)
11 Using the torso model, observe and discuss with colleagues:
• the extent of the breast (anterior to ribs 2‐6, extending from
the margin of the sternum to the midaxillary line)
• the posterior relations of the breast (pectoralis major, part of
serratus anterior and external oblique)
• the axillary tail of the breast. This may be felt by patients
and mistaken for enlarged axillary lymph nodes.
12 The mammary gland is removed from our specimens but you How does the internal structure
should refer to your lecture notes and models while discussing of the breast change with
the macroscopic structure of the breast with colleagues: puberty, pregnancy/lactation and
• nipple, areola menopause?
• mammary gland lobules, lactiferous ducts, lactiferous
sinuses
• fibrous septa (suspensory ligaments)
13 Identify the three main arteries that provide blood supply to the
breast:
• Lateral thoracic (a branch of axillary artery)
• Internal thoracic artery (perforating branches)
• Posterior intercostal arteries
14 Most of the veins draining the breast follow the arteries to end What is the course that might be
eventually in the internal thoracic and axillary veins. There is followed by metastases from the
some drainage into the posterior intercostal veins. The posterior breast to the vertebral column
intercostal veins are also connected (by the azygos system of and skull base?
veins) with the plexus of veins around the vertebral column
(called vertebral venous plexus); this connection could provide a
path for breast cancer to spread to the vertebrae. Identify the
posterior intercostal veins and the components of the azygos
system of veins (azygos and hemiazygos veins).

Materials: isolated thoracic vertebrae, parts of sternum, typical ribs, articulated skeleton, dissected specimens
of axilla, torso model, model of breast, thorax specimens with azygos venous system.
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Practical 2: Analysis of cell ageing
Principal Teacher: Dr. Louise Lutze‐Mann
Aims
To evaluate the genetic and environmental conditions that can influence ageing;
To produce and analyse growth curves for eukaryotic cells.

Introduction
Ageing and Werner’s Syndrome
The molecular mechanisms that underlie ageing are not yet well‐understood. Many different processes have
been implicated in the aging process, and it is likely that ageing is the result of a progressive accumulation of
changes. To date, the processes that appear to directly influence ageing, and so play a causal role in the ageing
process, are those involved in energy metabolism, oxidative stress and the maintenance of DNA stability.
Indeed, given the myriad of age‐related changes and the many proposed mechanistic theories of ageing, a
major problem in gerontology is distinguishing causes from effects (see Table 1, below).
Table 1. Genes that appear to modulate ageing in mammals
Gene name Common name Phenotype Primary reference
Mouse (Mus musculus)
GHR/BP Growth hormone receptor Increase in lifespan of 40–50% in Coschigano et al.
homozygous knock‐outs (2000)
IGF‐R1 Insulin‐like growth factor type Heterozygous mice live 26% Holzenberger et al.
1 receptor longer than wild‐type (2003)
MSRA Methionine sulfoxide Decreased lifespan and possible Moskovitz et al. (2001)
reductase A accelerated ageing in
homozygous knock‐outs
p53 Tumour suppressor protein 53 Heterozygous mutant mice Tyner et al. (2002)
display signs of accelerated
ageing
p66shc p66shc (SHC1) Roughly 30% increase in lifespan Migliaccio et al. (1999)
in ‐/‐ mice
PASG Proliferation associated SNF2‐ Possible accelerated ageing Sun et al. (2004)
like gene phenotype due to disruption
Pit1 Snell dwarf mouse Lifespan increase of 42% in Flurkey et al. (2001)
homozygous mice
Thdx1 Thioredoxin 35% increase in average Mitsui et al. (2002)
longevity in transgenic mice
XPD Xeroderma pigmentosum, Possible accelerated ageing de Boer et al. (2002)
group D phenotype due to homozygous
mutation

Human (Homo sapiens sapiens)
CKN1 Cockayne syndrome Type I Possible premature ageing due Henning et al. (1995)
to recessive mutation
WRN Werner syndrome Possible accelerated ageing due Yu et al. (1996)
to recessive mutation
Lamin A Hutchinson–Gilford syndrome Possible premature ageing due Eriksson et al. (2003)
to dominant mutation

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One of the most intriguing phenotypes in the biology of ageing is the accelerated ageing witnessed in humans
and animals as a result of certain mutations. Progeroid syndromes, as they are called, are rare genetic diseases
of which the two most impressive forms are Werner's syndrome (WS) and Hutchinson‐Gilford's syndrome. Both
these diseases have a phenotype that is remarkably similar to an accelerated ageing process, particularly in the
case of WS. Though differences exist in terms of pathology, what most markedly distinguishes these syndromes
is the age of onset, with Hutchinson‐Gilford's syndrome almost exclusively affecting children while WS patients
normally reach adulthood.

These diseases have attracted considerable attention because they may provide information about normal
ageing processes. The gene responsible for WS has been cloned and is designated WRN. The gene encodes a
member of the E. coli RecQ DNA helicase family that converts double‐stranded DNA into single stranded DNAs.
While it is known that E. coli RecQ participates in recombination and the repair of UV‐damaged DNA, the in vivo
functions of the eukaryotic RecQ‐like helicases are currently unknown.

Ageing and oxidative damage
Apart from inactivation of “longevity” genes, one of the prime candidates considered to contribute to ageing is
the accumulation of oxidative damage in cells. Eukaryotic cells are dependent on aerobic respiration for the
generation of the majority of their energy needs. This energy production (through the electron transport chain)
also generates a very small level of free oxygen radicals which can damage biological molecules. The
accumulation of this damage can lead to a decline in cellular function, the induction of mutations and,
eventually, the death of the cell. Cells contain a repertoire of compounds and enzymes such as ascorbate,
peroxidases and superoxide dismutases that can scavenge or inactivate the free radicals and so protect the cell.
Loss of these protective mechanisms can lead to the rapid accumulation of damage and cell senescence or
death, especially if the cells are stressed by high oxygen tensions (hyperoxia) or the presence of oxygen radical
generators such as hydrogen peroxide.

Use of yeast as a surrogate for human
The conservation of metabolic and signalling pathways between yeast and humans is strikingly high, leading to
the expectation that ageing mechanisms will also be common to both organisms. While this is not necessarily
the case, there are reasons to believe that in both organisms, similar cellular systems will fail earlier than
others. Certainly, many of the genes that extend yeast life span have human counterparts. Indeed, the WRN
gene that is mutated in Werner’s syndrome has a homologue, called SGS1, in the yeast S. cerevisiae. Yeast
mutants lacking SGS1 function have life spans only 40% that of congenic wild type strains and undergo what
appears to be a rapid ageing process. Initially, young sgs1 cells are indistinguishable from wild type by external
phenotypes. After about five divisions, sgs1 cells begin to slow down in their cell cycle and eventually become
senescent (cease dividing).
Similarly, yeast cells which have one of the superoxide dismutases (Mn‐SOD) inactivated (by insertional
deletion) are highly susceptible to oxidative damage and will rapidly accumulate damage which negatively
affects their lifespans. In this class, we will examine growth curves for the wild type yeast and for the sgs1 and
‐
Mn‐SOD mutants under different oxidant conditions.

Cell Culture
The ideal way to study factors affecting growth is to utilise cells that are growing in a defined and controlled
environment (tissue or cell culture). This can be achieved for many cell types, including those derived from
multicellular organisms, although there are limitations on the conclusions that can be drawn from experiments
conducted solely on cells cultured in vitro. While the controlled environment allows different conditions and
factors (drugs, growth factors, inhibitors) to be analysed, the complex interactions of cell types that occur in
vivo cannot be reproduced in the lab. Cell culture techniques have been established for many organisms from
both plants and animals as well as with various tissue types.

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A particularly useful experimental guidepost to the study of growth (and its decline!) involves the mathematical
prediction of growth rates. It can be demonstrated that simple equations that describe the colony growth of
microorganisms can also be used to describe growth of eukaryotic cells in cell culture. We will use these
equations to help discriminate different populations:
• a wild type yeast population growing under normoxia conditions;
• a wild type yeast population growing under hyperoxia conditions;
• yeast with a mutation in the Mn‐SOD gene, growing under hyperoxia conditions.
• yeast with a mutation in the SGS1 gene, growing under normoxia conditions;
Experimental (Work in pairs)
Innocula of the yeast were placed into growth media (YEPD: 10g/l yeast extract, 20g/l peptone, 20g/l glucose)
and allowed to grow with shaking (to provide aeration) for 18 hours at 30ºC under either normoxia conditions
or hyperoxia (95% O2, 5% CO2). Samples were removed at various times and stored on ice. Within a group you
need to analyse the growth of each of the four yeast samples (labelled A, B, C and D).

1. For each strain there are 8 samples that were collected at different times. Pipette 200 µl of each sample
into a well in a microtitre plate, making sure that the first well is a blank (YEPD media). You must swirl the
contents of the tube before you take your sample (why?).
2. Use the microtitre plate reader to determine the absorbance at 620nm (A620nm) for the samples. Transfer
these results to the table on page 5. Collect the results from another pair of students within your group so
that you can average the results.
3. You will need to determine the relationship between the number of cells in a sample and the absorbance
at 620nm that you measured. For this, count the number of cells in a defined volume. Use the samples
placed near the microscope for this part of the exercise. Each student should count one sample.
4. Pipette 20µl of yeast cells into a microfuge tube. Again, ensure that you swirl the tube containing the
yeast before you take the sample.
5. Add 20µl of Trypan Blue dye to your sample. Mix gently and thoroughly. Collect about 20µl with a
micropipette.
6. Immediately transfer the cell suspension to the notched side of the Kova Glastic Slide 10 by gently
pipetting the suspension into the chamber at the gap indicated by the arrow in the diagram below. You
will need to share the counting slide with other students as there are 10 counting chambers on each slide.

1. Transfer the slide to the microscope stage and focus on the grid on the slide using the 10X objective. Then
change to the 40X objective.
2. Count the number of cells within a small grid (an area that just fills the field of view when using the 40X
objective). Average your counts with those of the other pair of students sharing the slide.
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3. The section within the small grid contains a volume of 10‐5 ml. Therefore, the concentration can be calculated
from:
c = n/v
where c = cell concentration (cells/ml), n = number of cells counted, and
v = volume counted (ml). Use this to compute the concentration (cells/ml) for the yeast culture (don’t
forget the dilution factor!).
4. A linear relationship exists between the absorbance at 620nm and the concentration of cells in a sample
(until the number of cells becomes saturating and there are so many cells that the absorbance has
reached a maximum). Consequently, by extrapolation, you can determine the concentration of cells in
each of your samples based on their absorbance at 620nm. The sample that you counted was from a
yeast sample that had an absorbance of 0.3.
5. Use this data to complete the table on page 5 and then prepare a growth curve (cells/ml vs time) using
the graph paper provided. Collect the data for the other yeast populations (A, B, C and D) from the other
members of your group and plot the data for all samples on the one graph so that you can compare them
directly.

Time A620 A620 Average
Sample Cells/ml
sampled (hrs) Your data Colleague’s data A620
0
2
6
8
10
12
14
18

Data for the other samples from your group:
Time
Time
Sample sampled Cells/ml Sample Cells/ml
sampled (hrs)
(hrs)
0 0
2 2
6 6
8 8
10 10
12 12
14 14
18 18
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Time
Time
Sample sampled Cells/ml Sample Cells/ml
sampled (hrs)
(hrs)
0 0
2 2
6 6
8 8
10 10
12 12
14 14
18 18

6. Are the growth curves exponential in shape? If not, what shape are they? Why?
7. Label the following phases on your growth curves and identify what is occurring in each phase in the table
below:
Growth phase Growth characteristics
1 Lag phase
2 Exponential phase
3 Stationary phase
4 Declining phase

8. It is possible to determine the growth rate and generation time for these cultures using the following
fomula:
ln n = ln no + kt
where no is the initial number of cells in a culture, n is the number of cells after time t and k is a constant for that
culture representing the growth rate constant (growth rate/time). This follows ONLY for cells that are growing
exponentially. Consequently, you must choose values for no and n that are from the exponential phase of your
growth curve, with the appropriate time interval.

For example, if at 6 hours there are 1000 cells and at 12 hours there are 10,000 cells, then:
no = 1000; n = 10,000; t = 6 hours; and k can be calculated from:
ln 10,000 = ln 1,000 + 6k
9.2 = 6.9 + 6k
Therefore, k = 0.38/hr, or expressed another way, there is a 38% increase in population density every hour.

The generation time (g) i.e. the time for the population to double, can also be calculated, using the
following:
g = ln2/k
g = 0.69/0.38 = 1.82 hours
So the doubling time for this strain is 1 hour and 49 mins.

9. Calculate the growth rate and generation time for each of the yeast populations. Based on this
information and the shape of the growth curves, can you determine which population (A,B,C and D)
corresponds to each of the different growth conditions? Why did you reach this decision?
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Practical 3: Histopathology of Breast Lumps
Principal Teacher: A/Prof Gary Velan
Specific objectives
This practical class will provide you with an opportunity to recognise the histopathological features of common
causes of lumps in the breast. The emphasis will be on the importance of findings on histological examination
in distinguishing non‐neoplastic and benign lesions from carcinoma, as well as their relevance to assessing
prognosis.
Learning activities
Case 1 ‐ Clinical History
A 45 year old woman presented to her local medical officer with a lump in the right breast. The lesion, which
was in the lower outer quadrant of her breast, had been noticed to be enlarging over the last two weeks. There
was no change in the size or nature of the lump in relation to her menstrual cycle. On examination there was a
3 cm stony hard lump, which was tethered to the overlying skin. There was dimpling of the overlying skin on
contraction of the pectoralis muscle. There were firm, but mobile axillary lymph nodes on the right side.
Task 1
The class will be divided into 4 groups. Each group will be assigned one of the 4 virtual slides labelled
"Fibrocystic change, breast", "Fibroadenoma, breast", "Ductal carcinoma, breast" and "Metastatic
adenocarcinoma, lymph node". The members of the group should work together to prepare a presentation
based on their slide. The presentation should include a histopathological description of the lesion, followed by
a discussion of whether the lesion is consistent with the clinical features in this case. You may include
comment on risk factors and predisposing conditions, investigations, prognostic indicators and any other
relevant information. Each group will have 10 minutes to deliver their presentation; group members may elect
their own spokesperson(s).
Fibrocystic change, breast

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Fibroadenoma, breast

Ductal carcinoma, breast
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Metastatic adenocarcinoma, lymph node

Four months later the patient returned with back pain and right shoulder pain. She had lost 6 kg in weight over
the past two months and was anorexic. Examination revealed tenderness over the fifth and sixth thoracic
vertebrae and also over the right acromion.
Task 2
What is the likely cause of these symptoms and signs? What investigations would you perform to substantiate
your provisional diagnosis?

The results of biochemistry tests are outlined in the table below:
Clinical Chemistry
Analyte Measured Value Reference Interval
Sodium (mmol/L) 135 135‐145
Potassium (mmol/L) 4.1 3.4‐4.5
Chloride (mmol/L) 108 95‐110
Bicarbonate (mmol/L) 28 22‐32
Calcium (mmol/L) 3.0 2.10‐2.55 *
Phosphate (mmol/L) 1.9 0.8‐1.50 *
Urea (mmol/L) 6.0 3.0‐8.0
Creatinine (mmol/L) 0.09 0.05‐0.12
Bilirubin (mmol/L) 15 2‐20
Alkaline phosphatase (U/L) 285 38‐126 *
‐glutamyltransferase (U/L) 25 <30
AST (U/L) 31 <45
ALT (U/L) 26 <45
Tot Protein (g/L) 64 62‐80
Albumin (g/L) 35 33‐48
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Task 3
How would you interpret the abnormalities based on the patient's clinical features and her past history?

The patient died four months later. The virtual slide labelled "Metastatic adenocarcinoma, bone" was prepared
from tissues removed at autopsy. Your tutors will project and discuss this slide.
Task 4
Explain the sequence of events leading to the development of this lesion.

Homework Task
What are the likely causes of death in this woman? What other abnormalities might you expect to find at
autopsy?

References:
Kumar, V., Abbas, A.K., Fausto, N. & Mitchell, R.N. (2007). Robbins Basic Pathology. (8th ed.). Philadelphia, PA:
Elsevier Saunders.
• Chapter 6, pp. 173‐185
• Chapter 19, pp. 739‐750

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Practical 4: Cranial Cavity and Introduction to the Brain
Principal Teacher: Dr. Elizabeth Tancred
Specific objectives
1. Identify bones forming the cranial cavity and cranial fossae.
2. Identify meningeal coverings of the brain and major dural reflections (falx cerebri, tentorium cerebelli).
3. Identify major parts of the brain, including key gyri and sulci and lobes of the cerebral cortex, and
observe their relationship to skull bones, falx cerebri and tentorium cerebelli.
4. Identify the components of the ventricular system of the brain and its relationship to the subarachnoid
space
Learning activities
1. Identify the bones that form the cranial cavity – frontal bone (squamous and orbital parts), temporal
bone (squamous, petrous parts, mastoid and styloid processes), sphenoid bone (body, greater and lesser
wings) and occipital bone (squamous and basilar (clivus) parts, internal and external occipital
protuberances). Identify the sagittal, coronal and occipitotemporal sutures, the bregma and the lambda.
What is the significance of the bregma in infants?

2. Note the bones that form the anterior, middle and posterior cranial fossae and the hypophysial fossa.
Identify the following foramina – f. magnum, internal acoustic meatus, f. lacerum, f. ovale and f.
rotundum, jugular f. and the superior and inferior orbital fissures. As we proceed through the course
you will learn about the structures that pass through these foramina. You do not need to know their
details at this stage.

3. On prosected specimens examine the dura mater, the outermost layer of the meninges, which lines the
inside of the cranial cavity. Note the presence of tentorium cerebelli and the falx cerebri, double layers
of dura mater that divide the cranial cavity into compartments and hold the brain in position. On a brain
specimen identify the arachnoid mater and the pia mater. These are separated by the subarachnoid
space which contains cerebrospinal fluid and blood vessels.

4. Examine whole and half brain specimens and identify the components of the hindbrain – the medulla
oblongata (a continuation of the spinal cord), pons and cerebellum. Identify the relatively small
midbrain and in front of it, the hypothalamus and thalamus. Together the medulla, pons and midbrain
form the brainstem. The hypophysis (pituitary gland) attaches to the ventral surface of the
hypothalamus just behind the optic chiasm but it breaks off when the brain is removed from the skull.
In general terms what are the main functions of the brainstem, cerebellum and hypothalamus?

5. Identify the major parts of the cerebellum ‐ identify its two large cerebellar hemispheres, the relatively
narrow midline part that connects them, the vermis (L. vermis =worm), and the flocculus a separate
small area of cerebellar cortex on each side.

6. Now examine the large cerebral hemispheres, separated by the longitudinal fissure and covered by a
highly folded layer of grey matter called the cerebral cortex. The cerebral cortex has a very large surface
area and in order to fit into the cranial cavity it is thrown up into folds (known as gyri), separated by
grooves (known as sulci). On the lateral surface identify the central sulcus (passing downwards
approximately midway between the anterior and posterior poles of the brain) and the lateral sulcus.
Identify the precentral and postcentral gyri. On the medial surface identify the parieto‐occipital and
calcarine sulci. Now identify the four lobes of the cerebral cortex – the frontal lobe (anterior to the
central sulcus), the parietal lobe (between the parieto‐occipital and central sulci), the temporal lobe
(inferior to the lateral sulcus) and the occipital lobe (posterior to the parieto‐occipital sulcus). Note that
each lobe of the cerebral cortex is named according to the skull bone that overlies it. On the half brains
identify the corpus callosum, a large bundle of white matter that interconnects the two hemispheres.
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What part or lobe of the brain occupies each of the anterior, middle and posterior cranial fossae? What
is the function of the cerebral cortex (in general terms)?

7. Finally examine the main components of the ventricular system of the brain – the fourth ventricle
(between the pons and cerebellum), the cerebral aqueduct (passing through the midbrain), the third
ventricle (between the two thalami) and the interventricular foramen, an opening just in front of the
thalamus that leads into the lateral ventricle (located within the hemisphere). CSF is produced within
the ventricles. How does it get into the subarachnoid space?
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Practical 5: Brainstem
Principal Teacher: Dr. Elizabeth Tancred
Specific objectives
1. Identify external features of the medulla, pons and midbrain and understand their relationship to
internal structure.
2. Identify the boundaries and openings of the fourth ventricle, including the rhomboid fossa.
3. Identify the characteristic features of cross‐sections of the brain stem at the following levels: caudal
medulla, rostral medulla, mid‐pons, midbrain (superior colliculus) levels.
4. Identify major motor (corticospinal) and sensory (spinothalamic and medial lemniscus) tracts in the
brainstem.
Learning activities
Gross Anatomy
1. Study the external features of the brainstem. Inspect the medulla. Identify the pyramids, olive, gracile
and cuneate fasciculi and tubercles and the inferior cerebellar peduncle. The corticospinal fibres,
which run in the pyramids, cross to the opposite side at the junction of the medulla and spinal cord
forming the pyramidal decussation. At this level the ventral median fissure can usually be seen to
deviate to one side.
2. Inspect the pons. Identify the large middle cerebellar peduncle, the attachment of the trigeminal
nerve, the superior cerebellar peduncles and the superior medullary velum connecting them. The
ventral surface of the pons is formed by the base of the pons, which contains the pontine nuclei.
3. Inspect the floor of the fourth ventricle (rhomboid fossa). List the boundaries and openings of this
ventricle. Identify the obex, the most caudal point of the rhomboid fossa and the deep groove in the
midline, the median fissure, connecting the caudal end of the cerebral aqueduct with the entrance to
the central canal of the spinal cord. Further details of the rhomboid fossa will be studied in the next
practical.
4. Inspect the midbrain. Its dorsal part is formed by two grey masses, the superior and inferior colliculi,
which are collectively known as the tectum (L. = lid) of the midbrain. The ventral part of the midbrain is
dominated by the cerebral peduncles, two massive structures containing fibre systems descending from
the forebrain towards the brainstem and cord. Between them, ventrally, is the interpeduncular fossa.
Identify the exits of the two cranial nerves, which emerge from the midbrain, the oculomotor nerve
arising from the interpeduncular fossa, and the trochlear nerve arising from the dorsal surface, just
caudal to the inferior colliculus. Identify the isthmus, the narrow region of the brainstem that forms the
transition between pons and midbrain.
5. Examine the major arteries that supply the brainstem. Identify the paired vertebral arteries, which
unite at the pontomedullary junction to form the basilar artery. Identify the posterior inferior
cerebellar artery (arising from the vertebral) and superior cerebellar artery (from the basilar). The
basilar artery ends by dividing into the two posterior cerebral arteries. Note that each of these arteries
send small penetrating branches that enter the brainstem.

Microscopic Anatomy
Examine cross‐sections through the brainstem using “BrainStorm” on School computers. A CD of BrainStorm is
also available for purchase from the University Bookshop. Open the program, select Cross‐sections, select the
Brainstem submenu then select the ‘Caudal Medulla – Sensory Decussation’ cross‐section. The sections
photographed for these frames were stained for myelinated fibres, in which the tracts are darkly‐stained and
the grey matter appears lighter. The density of myelinated fibres within the grey matter is not uniform and thus
some nuclei (specific cell groups) within the grey matter are recognizable, as clear, less stained regions.

You will notice that the plane and level of the cross‐section are shown in the upper right hand corner of the
screen. Structures in the main image can be identified (selected) either by clicking on the structure in the image
or on its name in the Structures list. Once a structure is selected, a window with statement about the function
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of the structure can be displayed by clicking on the Function button and the structure can be followed through
consecutive levels by clicking on the up or down arrows in the upper right of the screen. You can view a gross
dissection, diagram or information screen about it by clicking on the G, D, I (or X – cross‐section) buttons.

As you identify the structures listed below on the computer sections, mark and label their location on the
outline diagrams provided.

1. Examine the Caudal Medulla – Sensory Decussation cross‐section. Note or identify the following
characteristic features:
(i) its roughly circular shape
(ii) the presence of the central canal, which extends throughout the spinal cord and into the caudal
medulla.
(iii) the pyramids, on the ventral surface – these are formed by the descending fibres of our major
motor tract, the corticospinal tract. (If you view the section below (click the down arrow in the
upper right corner) you will see that these fibres cross over to descend on the opposite side of
the spinal cord).
(iv) the gracile and cuneate nuclei form two swellings (the gracile and cuneate tubercles) on the
dorsal surface. These nuclei are part of a chain of neurons that transfer somatosensory
information from the spinal cord to the cerebral cortex. Fibres (axons) arising from cells in these
nuclei (internal arcuate fibres) can be seen passing ventrally and medially through the central
core of grey matter (the reticular formation) before they cross over in the sensory decussation.
They then form a prominent bundle called the medial lemniscus, which ascends through the
brainstem to the thalamus.
(v) the spinal nucleus of the trigeminal nerve – this is a continuation of the dorsal horn of the spinal
cord and is involved in relaying somatosensory information from the head to the cerebral cortex.

2. Now examine the Medulla ‐ Rostral cross‐section. (This can be reached by clicking the UP arrow three
times or by going back to the Cross‐Sections menu). The major distinguishing features of this level are:
(i) the presence of the very prominent inferior olivary nuclei, dorsolateral to the pyramids, (which
you will note, are still in the same location as the previous section).
(ii) the central canal is no longer present. Instead, it has opened out to form the fourth ventricle,
which is located dorsal to the medulla at this level. Note the choroid plexus, forming its roof.
What is the function of the choroid plexus?
(iv) the prominent medial lemnisci, lying on either side of the midline, dorsal to the pyramids
(v) the reticular formation, which, at this level, contains important respiratory and cardiovascular
control centres
(vi) the floor of the fourth ventricle is formed by nuclei associated with cranial nerves and which you
do not need to identify individually at this stage. However you will notice a sulcus called the
sulcus limitans, which separates nuclei involved in motor control (medial to it) from those
involved in sensory processing (lateral to it).
(vii) the inferior cerebellar peduncle, forming on the dorsolateral corner of the section.

3. Examine the Pons – Trigeminal Nuclei cross‐section. Note the following features:
(i) the two main parts of the pons, the large base (the basis pontis) and the relatively small
tegmentum more dorsally. The basis pontis contains grey matter (the pontine nuclei) scattered
amongst both descending and transverse fibres.
(ii) the corticospinal tracts passing down through the basis pontis
(iii) the massive middle cerebellar peduncles on its lateral sides. These contain millions of fibres
passing from the pontine nuclei into the cerebellum.
(iv) the fourth ventricle, dorsal to the pons, with the superior cerebellar peduncles forming its lateral
walls.
(v) the medial lemniscus, which has been pushed dorsally by the presence of the pontine nuclei and
associated fibres.

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4. Examine the Midbrain – Superior Colliculus cross‐section and note the following features:
(i) the tectum, forming the dorsal part of the midbrain. It is formed by two (paired) grey masses, the
superior colliculus at this level and the inferior colliculus, more caudally.
(ii) the cerebral peduncles forming the ventral part of the midbrain
(iii) the fourth ventricle is no longer present. It has closed down to form a rather narrow canal called
the cerebral aqueduct. This is surrounded by the periaqueductal grey matter.
(iv) the base of the cerebral peduncle (also known as the crus cerebri) is formed by white matter
which includes the corticospinal tract in its middle third.
(v) the decussation of the SCP (the dark mass in the centre) formed by the fibres of the superior
cerebellar peduncle (previously identified in the pons) crossing over to the opposite side
(decussating).
(vi) the substantia nigra, an important motor nucleus which will be studied later in this course.
(vii) the medial lemniscus, pushed laterally by the decussation of the SCP.

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Practical 6: Membrane Potentials
Principal teacher: Dr. Andrew Moorhouse
Safety Summary: Physiology Practical
MEMBRANE POTENTIALS
CLOSED SHOES REQUIRED
Hazards:
Chemical Hazard. Concentrated KCl can be an irritant
A. Safety controls to be observed:

Only dilute KCl solutions are used in the large reservoirs. The concentrated KCl
in the small reservoir should not need to be replaced unless it has become
contaminated. If you suspect this needs to be replaced, use gloves and carefully
use the pasteur pipette to refill the small reservoir.
B. In the event of an emergency evacuation:

• Stop the experiment, log off the computers and turn off the voltmeter
• Wash your hands.
• Dispose of gloves in the biological hazards bag
• Pack up your bags.
• Follow the instructions of the Demonstrators.
C. Clean-up:
• Dispose of gloves in the biological hazards bag
• Wash your hands thoroughly at the conclusion of the practical class.
Declaration:
I have read and understand the safety requirements for this practical
class and I will observe these requirements
Signature: ........................................ Date: .........................................

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INTRODUCTION
Students often find the concept of transmembrane potentials a difficult one. This class has been designed to
show that ‐‐ whatever the complexities of all of the fine details ‐‐ the principles which govern the origin and
magnitude of these potentials are relatively simple.

The first point to grasp is that the concentration differences which exist across the membranes of living cells –
i.e. between the intracellular fluid (ICF) and the extracellular fluid (ECF) ‐‐ are essentially energy differences.
When these fluids are partitioned by a membrane which allows no electrolyte permeation, then no potential
differences across the membrane occur. However, when the separating membrane is selectively permeable,
then ions move down their concentration gradient across the membrane and, as ions have positive or negative
charges, a transmembrane potential difference is established.

In this class we will model the cell with a simple Perspex apparatus which consists of two chambers (to
replicate the ICF and the ECF) separated by an artificial hydrocarbon membrane* which is selectively
permeable to either anions or cations. These ion exchange membranes have fixed charges within them (from
sulphonic acid or quarternary ammonium groups) that preferentially allow ions of the opposite charge to
permeate, and are used for various industrial applications (desalination, concentration of table salt, purification
of solutions).

KCl solutions of different concentrations will be used in the two chambers. One of these chambers will be
considered the ICF and its concentration will be unchanged at 150mmol/litre; the other will be deemed the ECF
and its concentration will be varied (as may occur in natural circumstances, though not to the extent which we
will employ in these experiments).

We have tried to minimise the technicalities. However, you should note that, in order to reduce artefacts
associated with the electrodes, they (AgCl‐coated silver [Ag] wire) are connected to the fluid chambers via
"salt‐bridges" (polythene tubes filled with 3 molar KCl in agar gel). The potentials will be measured with simple
digital multimeters (used as voltmeters). The experimental set‐up and procedures are given on the next page.

LEARNING OBJECTIVES
1) To measure potential differences across a selectively permeable membrane separating two solutions of
different ionic composition, and to measure how the magnitude of this potential varies as a function of the
ionic concentration gradient.

2) To determine if the artificial membrane is more permeable to cations (K+) or anions (Cl‐).

3) To calculate the theoretical equilibrium potentials for K+ and Cl‐ at different concentration gradients using
the Nernst equation and to consider how this relates to the measured potentials.

4) To gain experience plotting results and to consolidate principles relating to membrane potentials in
excitable cells

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Measurement of electrical potentials across an artificial membrane
The simple experimental setup used for measuring the transmembrane potentials is shown in Figure 1. Identify
and label the ICF, ECF, salt bridges, voltmeter and Ag/AgCl electrodes. Include the solution composition in the
different compartments.

Figure 1. The Experimental Set‐Up

The two halves of the perspex bath have been clamped together to sandwich an artificial membrane between
an aperture on each of them. The bath contains two main chambers (ICF and ECF) and two auxiliary recording
chambers (filled with 3M KCl) containing Ag/AgCl electrodes. Polythene salt bridges (with 3M KCl and 3‐4%
agar) connect the main chambers to the auxiliary chambers. This provides a stable method for measuring the
potentials, which are displayed on the voltmeter. The chamber connected to the voltmeter ground (ie zero
potential) electrode is defined as the ECF (just as membrane potentials are defined as the intracellular potential
with respect to the extracellular potential).

EXPERIMENTAL PROCEDURE
Fill both reservoirs with 150mmolar (mM) KCI (carefully making sure that there are no air bubbles in the vicinity
of the membrane) and ensure the salt‐bridges and silver‐silver chloride (Ag‐AgCl) electrodes are correctly in
place and the ECF chamber is connected to ground. Measure the potential (with the voltmeter set onto the
200 mV range). Consult a demonstrator if the value is not close to zero or is continuously changing. Record the
voltage. Note room temperature.

Keep the 150 mM solution in the ICF throughout the experiment. Use the Pasteur pipette to suck the solution
from the other reservoir (the ECF); make sure that all solution is drained from the tunnel which leads to the
membrane. Using a different pipette, fill this chamber with 100mmolar KCI solution; repeat the drainage
procedure and refill with the 100mmolar solution to ensure proper washout of the previous solution. Record
the voltage.
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Repeat the solution exchange procedure (double washout) and voltage measurement using the following
solutions: 50mmolar, 10mmolar, 5mmolar, 1.5mmolar. Flush the filling syringe with a little of each new
solution before adding it to the reservoir. Conclude with 150mmolar solutions in both chambers to check that,
in this experimental condition, the membrane potential has not shifted from its initial value.

EXPERIMENTAL RESULTS
i) Record the measured membrane potential values in Table 1 below.

ii) Based on your results, determine if your membrane is K+ selective or Cl‐ selective (think about the direction
of the KCl concentration gradient and the sign of the charge across the membrane)

proposed selectivity of the membrane ……………………………………………..

Table 1. Experimental and theoretical results
Concentration of KCl Membrane Potential Theoretical K+ Theoretical Cl‐
in the outside bath (mV) equilibrium (Nernst) equilibrium (Nernst)
[KCl]o (mM) Potential (mV) Potential (mV)
150 mM
100 mM
50 mM
10 mM
5 mM
1.5 mM
150 mM

Write some notes on how the membrane potential was generated. What ion was involved? What
direction did it move? Why did the voltage not continually change for the smaller gradients? Why
was there some drift in voltage for the largest gradient?

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iii) Calculate the theoretical equilibrium potential for both K+ (EK+) and Cl‐ (ECl‐) at each concentration gradient

using the Nernst equation (below). Include these results in Table 1. Use the space below to show each step in
at least one calculation of ECl‐ and one of EK+.

Recall that the Nernst equation gives the electrochemical equilibrium potential for a single ion. In other words,
it describes the membrane potential in the case of a membrane that is perfectly permeable to just one ion
species. As an ion diffuses across a membrane down its concentration gradient it carries a charge with it. This
charge difference, or electrical potential difference, will oppose further diffusion of the ions down their chemical
gradient. Very quickly the ion comes into equilibrium, where the electrical potential exactly balances the
chemical gradient, and there is no further net flux/movement of ions.
The Nernst equation is:
RT ⎛ [ X ]o ⎞
Vm = ⋅ ln ⎜⎜ ⎟⎟
zF ⎝ [ X ]i ⎠
where R is the gas constant (8.3145 VCmol‐1K‐1), T is absolute temperature (293.15 K at 20°C), F is Faraday’s
constant (96,485 Cmol‐1) and z is the valency of the ion species, X. The concentration of the ion, X, in the outside
bath is given by [X]o and that for the inside bath is [X]i.

ECl‐ calculation:

EK+ calculation:

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1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1
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iv) Plot your measured potentials against the concentration of KCl on both the standard (linear‐linear) graph
paper provided, and then on the log‐linear graph paper. Include a plot of either EK+ or ECl‐ against the K+ or Cl‐
concentration on your log‐linear graph paper.

v) Was each membrane potential equal to the theoretical Nernst potentials? What does it mean if the values
are exactly equal? What does it mean if the measured voltages are not exactly equal to the Nernst potentials
for K+ or Cl‐?

APPENDIX 1: REFINEMENTS USING ACTIVITIES RATHER THAN CONCENTRATIONS
Strictly speaking, quantification of all such phenomena which follow from the properties of electrolyte
solutions ought employ activity rather than concentration in every calculation. This is because inter‐ionic
attractions reduce the "freedom" of the ions to move and those attractions (and hence the difference between
calculated concentration and activity) are dependent upon ionic "strength" (i.e. concentration). Activity can be
calculated from concentration using the relationship:
a = γc
where: a is activity; c is concentration and γ is the activity co‐efficient (see Table, below).

c (mmol) 1.5 2.5 5.0 10 25 50 75 100 125 150
γ 0.958 0.946 0.927 0.902 0.858 0.817 0.750 0.770 0.753 0.740

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APPENDIX 2: CALCULATING THE MEMBRANE POTENTIAL WHEN BOTH K+ AND Cl‐ ARE PERMEABLE USING THE
GHK EQUATION

The Nernst equation is only equal to the membrane potential when only one species of ion can permeate the
membrane. When, as in this practical class, the membrane is permeant to more than one ion a more complex
equation, the Goldman‐Hodgkin‐Katz (GHK) equation, must be used to take into account the relative
permeability of the membrane to each ion present. When only K and Cl‐ are present, the equation can be
simplified using permeability ratios.

The simplified GHK equation is:
RT ⎛ PK : PCl .[ K + ] o + [Cl −]i ⎞

Vm = ⋅ ln ⎜⎜ ⎟⎟
F ⎝ PK : PCl .[ K + ]i + [Cl −]o ⎠
where Vm is the membrane potential, R is the gas constant (8.3145 VCmol‐1K‐1), T is absolute temperature
(293.15 K at 20°C), F is Faraday’s constant (96,485 Cmol‐1), PK : PCl. is the relative permeability of the
membrane to potassium and chloride, and subscripts o and I refer to the extracellular and intracellular
concentrations (or, even better, activities), respectively.
Results and other notes

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Practical 7: Oestrogen receptor binding
Principal Teacher: Dr. Anne Galea
Aims
• To illustrate the principles of a binding assay.
• To illustrate how the properties of oestrogen receptors present in breast cancer tissue might be
investigated.
Risk Assessment
• Diethylstilbesterol is carcinogenic.
• [3H]‐estradiol is radioactive.
• Lamb uterine tissue cell‐free extract is a potential biological hazard and potentially infectious.
• Gloves must be worn for this experiment and all laboratory work must be confined wherever possible to
an area of bench covered with disposable "bench‐coat".
• Demonstrators will give specific instructions on the handling of radioactive materials and these must be
adhered to at all times.
• All spillages must be wiped up immediately, and all waste placed in the containers for radioactive waste.

Introduction
The ability of a cell to respond to the hormone oestrogen depends on the presence of specific high affinity
receptors, since like other hormones it functions at very low concentrations (10‐8 ‐ 10‐10 M). Oestrogen
receptors are members of a superfamily of intracellular receptors that bind steroids, retinoids, thyroid
hormones and Vitamin D. When the ligand binds to the receptors, the receptors then bind directly to specific
regions in genomic DNA which regulate the transcription of specific genes. All members of the superfamily
have similar structures; they contain a transcription activating domain near the N terminus, followed by a DNA‐
binding domain with a hormone binding domain at the C terminal end of the protein. Binding of the hormone
changes the conformation of the receptor protein, exposing the DNA binding domain and promoting transport
3
to the nucleus. This practical involves investigating the binding of [ H]‐labelled oestrogen to its receptors in a
cell‐free extract of ovine uterine tissue.
Ligand/Receptor Binding Assays
The binding of a ligand to a receptor is a saturable phenomenon. The quantity of receptor is limited, resulting
in a finite number of binding sites and a limited capacity to bind a specific ligand. However, in crude systems
such as cell‐free extracts it is common to also observe a component that is non‐specific, has low‐affinity and is
apparently non‐saturable ligand binding. This results from interactions with a variety of components. Ligand
binding can be studied using labelled ligand, by incubating with a biological sample containing the receptor to
allow ligand‐receptor association to reach equilibrium, separating “free” (unbound) ligand from receptors (and
“bound” ligand) and measuring the amount of “bound” ligand associated with the receptors. It is essential that
the “label” should be measurable at very low concentrations and should not affect ligand‐receptor interactions.
3
As a result, radio‐labelling (e.g. [ H]‐oestrogen) is commonly employed for binding studies. Non‐specific ligand
binding can be assessed by carrying out parallel experiments in the presence of a high concentration of an
unlabelled ligand (e.g. diethylstilbesterol) for the receptor of interest. The unlabelled ligand saturates the
specific, high affinity receptor sites but does not affect low affinity non‐specific binding of the labelled ligand.
This is illustrated in the binding curves for [3H]‐oestrogen to rat uterine cytosol in the absence and presence of
unlabelled diethylstilbesterol (DES) shown below.
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[BOUND STEROID] (pmol)
1.0
Total
0.5 Specific
Non-Specific
0.0
0 5 10
TOTAL STEROID (nM)

Figure 1: Steroid binding to specific and non‐specific sites. The quantity of specifically bound steroid is
determined by subtracting non‐specific (+DES) from total (‐DES) binding.

The line designated as "total" (‐DES) in Figure 1, represents [3H]‐oestrogen bound to both specific receptor
sites and non‐specific binding sites and thus reflects both saturable and non‐saturable components. The
saturable, or receptor component is defined by the arithmetic difference between total and non‐specific
binding. Non‐specific or non‐saturable binding is measured as the [3H]‐steroid bound in the presence of excess
unlabelled competitive ligand (diethylstilbesterol). This method assumes that non‐specific binding sites are of
low affinity and high capacity relative to the receptor system, which is true for uterine oestrogen receptors,
and would have to be verified when investigating other hormone receptors.
Analysis of Binding Assays (Scatchard Analysis)
The method of choice for analysis of binding data (shown in Fig. 2) uses Scatchard Plots. This analysis permits
one to estimate the number of specific receptor sites (n) after subtracting non‐specific or non‐saturable binding
from total binding (as in Fig. 1) and the dissociation constant (Kd) of the ligand‐receptor complex (and thereby
assess the affinity of the receptor for the ligand).

All proteins, receptors included, bind their specific ligands (from the word “ligate”, to bind or attach) according
to the laws of mass action, i.e. there is an equilibrium between free receptor R, free ligand L (e.g. estradiol) and
the receptor‐ligand complex R‐L.

R + L <‐‐‐‐‐> R‐L

The dissociation constant Kd for estradiol and its receptor can be written as,

Kd = [R] [L] = ([R]total ‐ [R‐L] ) [L] ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 1.
[R‐L] [R‐L]

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This equation can be used to derive various forms of the Scatchard equation, of which the most useful for the
type of binding study in this experiment is:

B = (Bmax‐B) ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 2.
F Kd

where B = concentration of specifically bound ligand, F = concentration free ligand and Bmax is the total
concentration of ligand‐binding sites on receptors. Thus a graph of B/F against B (both of which can be
calculated from the experimental results) will give a straight line from which the Kd and the concentration of
receptor ligand‐binding sites on receptors can readily be determined ‐ see Figure 2.

3
B/F
0
0 1 2 3 4
B (nM)

Figure 2 Determination of receptor binding parameters. The data for specific binding is used for construction
of a Scatchard plot (B/F against B). The concentrations of “bound” (B) and “free” (F) ligand may be measured
directly in binding experiments. The Scatchard Plot gives a straight line with a slope of –1/Kd and an x‐intercept
of Bmax. Furthermore, the parameters Bmax (which equates to the number of receptor binding sites (n) present)
and Kd have practical use and significance. Consequently, Scatchard Plots are the preferred method for
analysing data from binding studies.
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EXPERIMENTAL (Work in Pairs)
Lamb Uterine Cell‐free Extract (prepared for the Class)
Tissue was homogenised in 10 volumes of 10 mM Tris/HCl buffer, pH 7.4, containing 1.5 mM EDTA and 5mM
sodium molybdate. Prior to homogenisation, 10 µl of 10% aqueous monothiolglycerol was added to 10 ml of
the above buffer. After centrifugation for 10 min at 600 x g to remove cell debris etc., the supernatant was
transferred to polypropylene tubes and centrifuged at 10,000 x g for 1 hour and the supernatant collected ‐ this
is the lamb uterine cell‐free extract.
Incubation with [3H]17β‐Estradiol (E2)
1) Set up the following twelve incubation mixtures in labelled microcentrifuge tubes:

Tube Number
Addition (µl) 1 2 3 4 5 6 7 8 9 10 11 12
Buffer 25 25 25 25 25 25 ‐ ‐ ‐ ‐ ‐ ‐
DES in buffer ‐ ‐ ‐ ‐ ‐ ‐ 25 25 25 25 25 25
Cell‐free extract 100 100 100 100 100 100 100 100 100 100 100 100
[3H]‐E2
0.75 nM 25 ‐ ‐ ‐ ‐ ‐ 25 ‐ ‐ ‐ ‐ ‐
1.5 nM ‐ 25 ‐ ‐ ‐ ‐ ‐ 25 ‐ ‐ ‐ ‐
3 nM ‐ ‐ 25 ‐ ‐ ‐ ‐ ‐ 25 ‐ ‐ ‐
6 nM ‐ ‐ ‐ 25 ‐ ‐ ‐ ‐ ‐ 25 ‐ ‐
12 nM ‐ ‐ ‐ ‐ 25 ‐ ‐ ‐ ‐ ‐ 25 ‐
30 nM ‐ ‐ ‐ ‐ ‐ 25 ‐ ‐ ‐ ‐ ‐ 25

Buffer: 10 mM Tris/HCl, 1.5mM EDTA, 5mM sodium molybdate, pH7.4.
DES: 1.5 mM diethylstilbesterol in the above buffer.
[3H]‐E2: [2,4,6,7‐3H]17β‐estradiol.

2) Carefully mix the contents of each tube by inversion.
3) Place in 5˚C water bath for at least 30 min. While the tubes are incubating, discuss the experiment
with your demonstrator.
4) At the end of the incubation, centrifuge briefly (why?) before adding 150 µl dextran‐coated charcoal
to each tube and mixing.
5) Transfer tubes to the 5˚C bath and mix four or five times over a 5 min. period.
6) Centrifuge for 2 min.
7) Carefully remove tubes. Place your tubes in a rack and transfer 150 µl of clear supernatant into clean,
labelled microcentrifuge tubes (numbered 1‐12). Do not disturb the charcoal which contains the
unbound [3H]E2. The clear supernatant contains the protein‐bound [3H]E2. Place your 12 tubes in the
rack provided by your demonstrator. Your samples will be processed overnight and the average
group data for dpm in each of the 12 tubes will be provided online within the next week.

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RESULTS and CALCULATIONS
The specific activity of the [3H]17β‐Estradiol is 61.8 Ci/mmol. (This means that 1 nmol of the [3H]17β‐Estradiol
gives 1.36 x 108 dpm. The protein concentration of the lamb uterine cell‐free extract is 0.60 mg/ml.

[3H]17β‐Estradiol concentrations
Determine the Total [3H]‐E2 concentration in each binding assay tube. The concentration of [3H]‐E2 added to
tubes 1 and 7 was 0.75 nM. However, this was diluted by the addition of 100 µl cell‐free extract and 25 µl
buffer or DES in buffer. Calculate the final total concentration of [3H]‐E2 in the 150 µl binding assay. Repeat
this calculation for the other 10 tubes.

Multiply your average group dpm values for each tube by 2 (only 150 µl of the 300 µl after addition of charcoal
was measured). Use the specific activity given above to calculate pmol of bound estradiol (both total and non‐
specific). Calculate pmol of specifically‐bound estradiol by subtraction, and plot your data as shown in Figure 1.

Calculate the concentrations of specifically‐bound (B) and non‐specifically‐bound estradiol at each total
estradiol concentration from the pmol values calculated above. Remember that the total volume in each
binding experiment was 150 µl (the volume before addition of the charcoal).

Calculate the concentration of free estradiol (F) at each total estradiol concentration by subtracting the
concentrations of both specifically‐ and non‐specifically‐bound estradiol from the totals.

Scatchard Plot
Calculate values of B/F and plot your group data as a Scatchard Plot (see Figure 2). From the Sctachard Plot
calculate the Kd of the Oestrogen Receptor for estradiol and the concentration of estradiol binding sites in the
binding assays (=Bmax). Using the protein concentration of the cell‐free extract, calculate the concentration of
estradiol binding sites per mg protein.

Sample data: Tube No. dpm
1 519
2 1031
3 1479
4 2533
← These are data obtained by
5 2976
the laboratory staff in setting
6 4729
up the experiment.
7 26
8 50
9 109
10 196
11 385
12 1076

Sample Calculations
Your calculations might look like this. (The "Sample data" for tubes 1 & 7 have been calculated.)
Tubes [E2] [E2] Total bound Non‐spec bound Specific bound [B] nM [F] nM [B]/[F]
(stock) nM (assay) nM E2 (pmol) E2 (pmol) E2 (pmol)
1 & 7 0.75 0.125 0.00763 0.00038 0.00725 0.0483 0.0741 0.652
2 & 8 1.5
3 & 9 3
4 & 10 6
5 & 11 12
6 & 12 30
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Practical 8: QMP: Weighing the Evidence
Principal Teacher: Dr Rachel Thompson

Students should prepare for these classes in the following ways:

1st years >>> should begin the QMP online tutorial 5 on Critical Appraisal and go on to the tutorial 6 on Bias
and Trial methodology if you have time. Bring any questions you have on these topics with you to the class.

2nd years >>> should revise both of these tutorials with attention to the "Advanced Student" areas. Come
ready to do a prac (will be working in small groups)

Bias in trials
Aims of the practical:
To consolidate the knowledge and skills learned in the QMP Online tutorials 5 and 6.
This practical aims to start first years on the road to understanding critical appraisal and the problems that
occur in trial methodology. For second years, there will be some revision prior but a new prac based on
learning more about the detection of bias.
This practical aims to give you the opportunity to study some papers on trials and work through set questions
on bias within a supported environment.

Learning objectives
• To consolidate what you have learnt so far about bias in medical trials
• To improve critical thinking skills
• To work within pairs and small groups

PAPER 1: Hip Protector Study
Kannus, P. et al. (2000). Prevention of hip fracture in elderly people with use of a hip protector. NEJM, 343,
1506‐13.

Instructions:
• First read the paper thoroughly. Specifically look for major forms of bias and *ANTI‐BIAS* in the paper.
Think about the stages that you went through to design your trial in SGS4.
• Discuss the paper with your partner or group and then answer the following questions.
• Answers will be available online in a week but try and work it out first.
• Tutors are available to answer your queries.

Relative Hazard (using Cox’s Proportional Hazards and Poisson analysis) is used for analysis of most of the
results here. These are statistical methods that can test for significance and are useful where there is significant
expected loss in the trial due to death and other outcomes (e.g. here with people leaving the trial as they fall
and fracture their hip). It is often used in trials where the main outcome is death such as in oncology
treatment. The Hazard Ratio is like relative risk and is used when the risk of developing the outcome in
question is not constant over the time of the trial. By measuring the risk over time it will take into account the
loss by attrition as subjects developing the outcome can no longer continue in the trial. If the hazard ratio (HR)
is 0.5 then the relative risk of dying in one group is half the risk of dying in the other group. The outcomes are
often shown graphically as you will see in this paper.

nd
Advanced students (i.e. 2 years and the mathematically minded) may wish to read : Spruance et al. (2004).
Hazard Ratio in Clinical Trials. Antimicrobial Agents and Chemotherapy, 48, 8, 2787‐2792: Accessed 05.9.10:
http://aac.asm.org/cgi/content/full/48/8/2787
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1. Trial population:
Who were the subjects and how were they enrolled? What bias might this cause?

How might the researcher’s choice of subjects right at the beginning of a trial affect the external validity of
this trial?

2. Selection Criteria:
Can you list the selection criteria used for this trial?

Do you see any problems with these that might affect the internal or external validity of the trial?

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3. Baseline assessment:
Did the researchers show the baseline assessment?

What does this show and how do you think this might affect the trial?

4. Trial groups:
Why do you think they decided to have ‘treatment’ units and ‘control’ units (p.1507)?

How might this choice of allocating subjects for the intervention and control groups affect the results of
the trial?

What bias is this and was there another way around the original problem that might have avoided this?

5. Group allocation
How did the researchers allocate the numbers between the groups and do they explain why they do this?

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6. Randomisation:
Was the randomisation method explained fully and what can you look at to see whether it was done well?

Was it done well here? If not – why not?

7. Confounders:
Can you think of any possible confounding factors that might affect the validity of this trial?

Did the researchers mention any of these specifically?

What did they do to minimise their effect?

8. Trial intervention:
What was done to minimise bias during the running of the trial?

What was not done that could have been done? What bias would this minimise?

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9. Drop‐outs:
How many subjects were lost during the follow‐up?

Should we include those who refused in our analysis as they did not take part in the trial at all?

Did the authors look at this and did they compare the two groups’ base‐line characteristics before or after
the refusers left?

What might be important about these two groups of refusers?

How did the researchers deal with the attrition effect?

Did they do enough? If not – what should they have done and what difference might this have made to
their results and conclusion?

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10. Conclusions:
What are you overall impressions of this paper and the trial?
Was the trial well‐conducted? Are there major flaws due to bias? If there are – what would you have done
to make it better?

Was the paper itself well‐written? If it wasn’t – what would you have done?

Next

When you have finished the prac worksheet you should go on to analyse this trial using the Critical Appraisal
Worksheet introduced in the lecture (week 2). The worksheet is available in Blackboard.

Some of the answers will be much easier now as you have taken such a close look at the biases involved in this
trial.
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Practical 9: Cranial Nerves
Principal teacher: Dr. Elizabeth Tancred
Specific objectives
1. To understand the fibre composition of each of the cranial nerves 3 ‐ 12.
2. To review the origin of cranial nerves 3 ‐ 12 from the brainstem.
3. To review, on the skull and on dissected specimens, the site where each nerve pierces the dura mater
and the point of emergence from the skull of each cranial nerve.
4. To review the distribution and functional significance of cranial nerves 3 ‐ 12.

Learning activities
1. On whole brain specimens and brainstem models identify each of the following cranial nerves:

Oculomotor n. (CN 3) – emerging from the interpeduncular fossa before it passes between the superior
cerebellar and posterior cerebral arteries.

Trochlear n. (CN 4) – emerges from the dorsal surface of the midbrain, just below the inferior colliculus
and then winds around the edge of the cerebral peduncle to reach the ventral surface. (Because of its
size and fragility this nerve is often difficult to see in specimens.)

Trigeminal n. (CN 5) – the largest of the cranial nerves, emerges from the middle cerebellar peduncle at
the side of the pons.

Abducens (CN 6), facial (CN 7) and vestibulocochlear (CN 8) nerves – emerging (from medial to lateral)
along the pontomedullary junction.

Glossopharyngeal (CN 9) and vagus (CN 10) nerves, emerging from medulla along the lateral surface of
the olive.

Accessory n. (CN 11) – emerging in a line extending down the ventral surface of the medulla to the
upper levels of the cervical spinal cord.

Hypoglossal n. (CN 12) – emerging as rootlets from the medial surface of the olive.

2. On deep head dissections identify where each of the above nerves pierces the dura mater. On
dissections of the cavernous sinus, identify the trigeminal ganglion and its branches, the ophthalmic,
maxillary and mandibular nerves. What is the function of the trigeminal ganglion? On dried skulls
identify the following foramina – the superior orbital fissure, optic canal, foramen rotundum, foramen
ovale, foramen lacerum, jugular foramen, internal acoustic meatus, hypoglossal canal and
stylomastoid foramen. List the major structures pass through each of these foramina.

3. Review the function of the oculomotor, trochlear and abducens nerves. On orbit models identify the
oculomotor, trochlear and abducens nerves and their branches (although the branches do not need to
be named). Identify the ciliary ganglion, the levator palpabrae superioris muscle and the extraocular
muscles (as a group). (It is not necessary to identify each muscle by name at this stage – this will form
part of a presentation by students in SGS 8).

4. Review the general functions of the trigeminal n. and its branches. In specimens and orbit models
identify the ophthalmic and maxillary nn. Find the terminal part of the maxillary n. (the infraorbital n.)
as it emerges onto the face (through the infraorbital f.). Identify the mandibular nerve (emerging from
the mandibular foramen) and its inferior alveolar and lingual branches. What do they supply? Identify
the temperomandibular joint and the muscles of mastication (as a group). Because the nerve arises
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behind these muscles many of its branches to them have been removed during dissection. How would
you test the integrity of the trigeminal n.? Map the location of the sensory deficits resulting from
lesions of the ophthalmic, maxillary and mandibular branches.

5. In specimens of the face and head identify the parotid gland and the muscles of facial expression (as a
group). Identify the facial n. at the stylomastoid f., where it emerges from the skull before entering the
parotid gland. Note the distribution of its branches, which can be seen passing onto the face from the
parotid gland. Other than the muscles of facial expression, what else does the facial nerve supply?

6. Review the functions of the vagus n. (which was studied previously in a Year 1). Identify this nerve in
neck pro‐sections and observe its relationship to the internal carotid artery and internal jugular vein.

7. Identify the hypoglossal nerve in the neck and follow its course to the tongue muscles, which it
supplies.

Note:
BrainStorm contains extensive text screens, gross images and diagrams of the cranial nerves, including a
simulated cranial nerve exam, with which the effects of lesions of individual nerves can be studied. Because of
time constraints the effects of lesions of the cranial nerves will not be covered in this practical. Instead they
will be the focus of clinical case presentations in SGS 8. Students in this course are not expected to study
details of the nuclei associated with each cranial nerve, nor do they need to name the branches of the nerves
except those mentioned above. They should however have an understanding of the composition and
functions of each nerve.

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Practical 10: Somato‐sensory System
Principal teacher: Dr. Richard Vickery
Introduction:
The somatosensory system is a complex sensory system involving receptors from several sensory modalities:
touch, temperature, pain, and proprioception/kinaesthesia. The receptor systems are quite distinct but all are
represented in cortical areas 3a & 3b (S1) as well as other brain areas. Beyond primary sensory cortex lie
specialised processing areas for each sense, and then integrative cross‐modal areas where the different senses
converge.

The experiments in this practical class are designed to help you learn about the cerebral cortex and the way
that the cortex perceives somatosensory stimuli. The primary focus of the practical class is not on the
properties of the peripheral receptors, but rather on the integration of information, including from multiple
senses, and the way that integration can be manipulated to alter perception.
Aims:
1. Be able to explain the contribution of slowly and rapidly adapting receptors to tactile perception.
2. Be able to explain the role of spindle afferents in proprioception, and the contribution of the
vestibular, vision and proprioceptive senses to balance.
3. Be able to explain sensory convergence and describe an example.
4. Be able to describe the topographic map of tactile inputs in primary somatosensory cortex, and how
this map is subject to ongoing modification.
1. Tactile Perception ‐ role of receptor types and motor system (efference copies)
Active versus passive texture discrimination
Tactile sensation is an active sense, in part because of interactions with the motor system, and also due to the
many rapidly adapting receptors. We will examine both of these features and should be able to show that
restricting tactile activity to only slowly adapting receptors significantly reduces the performance of the tactile
system.

1) For this experiment your subject must be blindfolded. The subject has to discriminate the difference in
texture between two squares of sandpaper. We will test them under several different conditions.
2) You should have a set of sandpaper squares with grit #'s as indicated on the table below. We will use 240
as a standard. The subject will be asked to feel the standard and then a different square of sandpaper and
should then rate the second square as “rougher” or “smoother”.
3) For the Active touch condition the subject should be guided to each sandpaper square, and then she is
allowed to explore the surface for ~2s before having her finger to the second square where she again has
~2s to explore the square before making a judgement.
4) Present the second squares in a random order. You will need to test each one 8 times to get useful data.
5) Now choose whether to test the role of the motor system by testing Passive Touch or the role of rapidly
adapting receptors by testing Static Touch.
6) For Static Touch, take the subject's index finger and rest the tip on each of the squares of sandpaper in
turn, beginning with the reference sheet. The subject must not move their finger so as to reduce the
amount of rapidly adapting tactile activity. As before the subject must report whether the second sheet of
sandpaper is “rougher” or “smoother” than the standard.
7) For Passive Touch, take the subject's index finger and move the tip backwards and forwards on each of the
squares of sandpaper in turn, beginning with the reference sheet. The subject must relax their hand and
not make active movements, but let the experimenter control their movements. As before the subject
must report whether the second sheet of sandpaper is “rougher” or “smoother” than the standard. .
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Grit # Rep. 1 Rep. 2 Rep. 3 Rep. 4 Rep. 5 Rep. 6 Rep. 7 Rep. 8 % called
rougher
Active touch
60
100
180
240
320
360
400
Static touch
60
100
180
240
320
360
400
Passive touch
60
100
180
240
320
360
400

How different does the coarseness (grit number) have to be before the subject can reliably discriminate between
them using the active touch protocol?

Does the accuracy of discrimination change when the subject can move only passively, or cannot move at all?
Why?

What additional information would be required to build a mental map of the textures and objects on a table in
front of you when exploring blindfolded with just your finger tips?

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2. Kinaesthetic Sensations
Kinaesthesia (perception of movement) and proprioception (perception of position) both utilise a variety of
somatosensory receptors: joint receptors, Golgi tendon organs, and muscle spindle receptors. Some of these
receptors, in particular muscle spindles, are exquisitely dynamically sensitive, and can signal both muscle
length and rate of change of muscle length. We can exploit this sensitivity to explore kinaesthesia by using a
vibrator applied to a tendon: the small but rapid changes in muscle length cause strong activation of the
spindle afferents.
2.1 The role of muscle spindles in proprioception
We will explore the contribution of muscle and joint afferents to the sense of limb position
1. Blindfold your subject and have him rest his elbows on the bench while holding his arms in front of his
body in a relaxed manner (half‐way between full flexion and extension).
2. Now grasp one forearm and move the arm around the elbow (bend it up and down) and ask the
subject to follow the imposed movement with his other arm so that their positions match.
3. Now repeat the procedure while you apply a vibrator to the biceps tendon of one arm.

Does the vibration cause errors in the movement?

What might cause the errors?

• Repeat the procedure with the subject’s blindfold removed. Are the movements more accurate?

What information is being used in making these movements in the two cases?

What does that tell you about the relationship between the senses, in terms of which tends to dominate
perception?

Try another demonstration of the same phenomenon is to have the subject stand still and close their eyes.
1. Ask the subject to SLOWLY reach towards his nose with the unstimulated arm so as to touch the tip of
his nose with his index finger (this should pose no problem).
2. Now apply the vibrator to the tendon of the triceps muscle. Ask the subject to repeat the action of
trying to slowly try to touch his nose with the stimulated arm.

Can you explain the subject's actions/perception on the basis of your knowledge of the muscle spindle afferents
and their role in proprioception?

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2.2 Maintaining posture ‐ the role of three different sensory systems
Staying balanced upright on just two legs is an essential requirement for normal human function. We use three
different sensory systems to assist us in this task. In this experiment we evaluate their respective contributions.

1. Ask the subject to take his/her shoes off, put both feet together and stand straight with eyes closed.
2. The demonstrator now applies a vibrator to each Achilles tendon. For safety, a classmate should stand
behind the subject to prevent them from falling.
3. Repeat this procedure with the subject’s back in contact with the bench top.
4. Finally, repeat the procedure with the subject’s eyes open.

What did the subject experience/do in the first case?

What additional information was added in the second experiment, and did it affect the subject's experience?

What further information was added in the third case and did it change the subject's experience?

3. Cross‐modal convergence in sensation
3.1 Parchment skin illusion
In this experiment we will examine the role of convergent auditory inputs in shaping tactile perception.
1. Put the headphones over your ears and adjust the volume to a comfortable level, then close your
eyes.
2. With the tone control set to bass, rub your hands together gently about 10cm in front of the
microphone so you can hear the sound of them rubbing. If you can't hear the sound clearly, then try
directly rubbing the edge of the microphone.
3. Note whether your hands (or the microphone edge) feels smooth or rough.
4. Now have a friend adjust the tone control to a high treble component, and then repeat the rubbing
movements,and again note the apparent roughness.

Does your perception of skin texture change when you change the auditory feedback? If so, how?

Where in the nervous system is this change occurring?

What does this tell you about the relationship between hearing and touch? What does it tell you about
integration between the senses?

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3.2 Wet hands illusion
In this experiment we will examine the role of convergent somatosensory inputs in shaping tactile perception.
1. Put on a surgical glove of the appropriate size.
2. Put your hand under cold running water in the sink at the front of the lab. What do you feel?
3. Now put your hand in the bucket of warm water. What do you feel?
4. If you did not have a strong sense of wetness under the cold tap, have one of your lab partners cut
slits in two glove fingers (without you knowing which fingers have the slits). Then try under the cold
tap again and see whether you can identify which fingers have got the slits (and so are truly wet).

Do we have somatosensory receptors for the stimulus of “wet”?

By comparing the two sensations, can you determine what gives rise to the sense of “wetness”?

At what level of the nervous system does this illusion occur?

4. Organization of cortical maps
4.1 Archimedes’ illusion
Vision and touch both use topographic organisation where the sheet of receptors in the periphery (retina or
skin) are connected in a smooth map‐like fashion through to sheet of cortical neurons in the primary sensory
cortex. In this experiment we can probing the cortical organization and interpretation strategies for the sense
of touch.

1. Cross your index and middle fingers over each other so that the tips are a centimetre apart.
2. Now close your eyes and try and roll a small bead back and forth between the tips of these fingers.
3. What happens as the bead rolls up and down the gap, or from one finger to another?

Does it feel like one stimulus or two?

If you un‐cross your fingers and touch the same parts, does it feel like one stimulus or two?

If they seemed different, what explains the difference in perception? Where would the re‐interpretation occur?
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4.2 Whose hand is that?
The cortical map has its broad parameters fixed in development, but is constantly updated by day to day
experience. We will demonstrate a remapping of proprioception based on congruent visual and tactile
stimulation.
1. Have your subject put on their lab coat with only one arm in its sleeve. They should then sit with both
their arms palm up on the table. Put the rubber arm in the empty sleeve, and position next to the real
arm (real arm on the outside). Use the folder to screen the real arm so that the subject can not see it.
2. While the subject watches, use the cotton tips to touch the rubber hand and the subject’s own
matching hand (hidden by the screeen) in exactly the same way – i.e. if you brush along the index
finger of the rubber hand, do the same thing to the subject’s hand simultaneously. You can alternate
with occasional stroking of the real unpaired hand.
3. Keep this up for several minutes, and after a while the subject may begin to identify the rubber hand
as part of their own body.
4. Test the 'adoption' of the rubber hand by stroking just the rubber hand without a matching stimulus of
the normal hand. The subject may report a feeling of “numbness”. A more drastic test is to try hitting
the rubber hand with an object and observing the subject to see if they try to withdraw it!

Why might the subject identify the hand as their own?

What is happening during the time that the experimenter is stimulating the real hand and the rubber hand?
Where is it likely to be happening?

What utility does this remapping have in normal life?

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Practical 11: CNS: Normal and abnormal
Principal teachers: Patrick de Permentier and Assoc. Prof. Gary Velan
Learning objectives
1) Obtain an understanding of the normal histological appearance of selected central and peripheral
nervous system tissues namely spinal cord, cerebellum and peripheral nerve.
2) To examine unique microscopic characteristics of each of the nervous tissues.
3) To introduce the histology and neuropathology associated with cerebral infarction and haemorrhage.
Nervous Tissue
A brief description of Nervous Tissue
The brain and spinal cord comprise the central nervous system (CNS). The nerves that emerge from the spinal
cord and brain to pass to parts of the body are the peripheral nervous tissue (PNS). Nervous tissue, with many
interconnections, forms a complex system of neuronal communication within the body and is specialized for
detecting stimuli, integrating functions, controlling effectors and higher functions. Nervous tissue consists of
cell bodies, cell processes (nerves), and neuroglia (supporting cells).

Cell Types
Neurons: Structural and Functional units of the nervous system
These cells (around 12 billion) are responsible for the receptive, integrative, and motor functions of the
nervous system. They can generate nerve impulses (irritability), and can transmit these impulses along their
processes (conductivity). They range in diameter from 5 to 150 µm and contain 3 parts: a cell body, multiple
dendrites and a single axon.

1) Cell body (soma, perikaryon) is the region of the neuron containing a large pale‐staining spherical,
nucleus with a conspicuous nucleolus and perinuclear cytoplasm.
2) Dendrites project from the cell body and are specialized for receiving (afferent) stimuli from sensory cells,
axons and other neurons which are then transmitted towards the soma.
3) Axons arise as a single thin process extending longer distances from the cell body than the dendrite. As
with dendrites, the terminals of the axon are branching and terminate in end bulbs (terminal boutons),
which come close to another cell and form a synapse.

Peripheral Nerve Fibers
Peripheral nerves are bundles (fascicles) of nerve fibers (axons) surrounded by several CT sheaths. Each bundle
contains sensory and motor components.

Myelinated Fibers (1‐20µm diameter)

Myelin (rich in lipid) is the membrane of the Schwann cell organized into a spiral sheath that is wrapped
several times around the axon. Schwann cells are cells whose cytoplasm contains a flattened nucleus, a small
Golgi apparatus, and a few mitochondria. Myelinated fibers are capable of rapid transfer of impulses (touch
sensory pathways).

Unmyelinated Fibers (less than 2µm in diameter)

Some axons in the PNS are surrounded by Schwann cells but not wrapped with layers of myelin. They are
found in pain and temperature sensory pathways and motor paths to the viscera.
Virtual Slide Box
The virtual histology slides for this and subsequent practicals can be found at:
http://vslides.unsw.edu.au/VirtualSlideV2.nsf/id/8074FA
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Additional virtual images can be found on the student computers by accessing Class Program‐School of
Medical Sciences, UNSW. Then click on Anatomy followed by Neocortex Virtual Microscope‐Histology‐P.
Groscurth (Zurich, Web)

Learning activities
Spinal Cord
Virtual Slide Box (Spinal cord and Spinal cord smear) and Zurich Virtual Slide database (Spinal cord‐Thoracic
Segment‐Luxol Fast Blue, Neutral Red and Spinal cord‐Lumbar Segment‐Azan and Meninges‐Azan).

Identify gray and white matter, central canal (surrounded by ependymal cells), dorsal and ventral horns,
meninges (pia, arachnoid and dura mater), subarachnoid space with dorsal and ventral rootlets, blood vessels,
a motor neurone with a cell body (soma), nucleus, nucleolus, Nissl granules, an axon with axon hillock area,
dendrites, glial cells (oligodendrocytes, astrocytes).

• What is the difference between white and gray matter?
• What is the function of ependymal cells?
• What do Nissl granules represent?
• What function do the meninges serve and what type of tissue are the meninges made up of?
• What is the functional difference between an axon and a dendrite?
• What is the function of an oligodendrocyte and of an astrocyte?
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Cerebellum
Virtual Slide Box (Brain/Cerebellum and Cerebellum silver stain) and Zurich Virtual Slide database (Cerebellum
silver stain).

• Identify the folia (folds), meninges (pia and arachnoid mater), blood vessels, and white and gray matter.
The gray matter is subdivided into 3 distinct layers namely outer molecular, inner granular and middle
Purkinje cell layer. Note the processes on the Purkinje cells.
• What does white matter consist of?
• What is the function of the Purkinje fibers?

Peripheral Nerve
Virtual Slide Box (Peripheral Nerve) and Zurich Virtual Slide database (Nerve; Goldner and Nerve;
Haematoxylin and Eosin)
• Identify fascicles (bundles) of nerves, levels of connective tissue wrappings (epineurium, perineurium,
endoneurium), fibroblast nuclei, adipose tissue, blood vessels, myelinated nerve fibers, axons, and
Schwann cells.
• What is the function of a Schwann cell and what effect does myelin have on nerve transmission?
• Why do 3 levels of connective tissue wrap nerve fibers?

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Neuropathology Component
Case History
A 56‐year‐old man was admitted to hospital through the Casualty department after an hour‐long episode of
dull, central chest pain at rest accompanied by shortness of breath and profuse sweating.

He was investigated and treated for myocardial infarction, and appeared to be slowly recovering until 6 days
later, when he developed right‐sided hemiparesis and severe aphasia. His level of consciousness deteriorated,
and he died 3 weeks later.

Task 1
A virtual slide of a cerebral infarct was prepared from tissues removed at autopsy. Examine the virtual slide,
which has 3 distinct areas – a zone of liquefactive necrosis, a zone of reactive gliosis (healing) and a zone of
normal cerebral cortex. An Adaptive Tutorial is available to assist with identification of these regions.

Write brief descriptions of each of these areas. In which vascular territory did this lesion occur? Could this
lesion have been responsible for the patient's death?

Task 2
Examine the virtual slide of a cerebral arteriovenous malformation, which is a congenital abnormality of blood
vessels within the cranial cavity.

What types of vessels can you observe?

What are the likely clinical effects and complications of such a lesion?

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Additional resources to support this practical and help with revision
Prescribed Textbook:
• Junqueira, L.C. (2010) Chapter 9 in Basic Histology, (12th ed.). New York: Lange Medical Books/McGraw‐
Hill
• Frosch, M.P. in: Kumar, V., Abbas, A.K., Fausto, N. & Mitchell, R.N. (2007). Chapter 23 in Robbins Basic
Pathology, (8th ed., pp. 860‐869.). Philadelphia, PA: Elsevier Saunders.
Computer resources
1. LANGE Educational Library, the medical education resource for on‐going study, review, and reference.
http://info.library.unsw.edu.au/cgi‐
bin/local/access/access.cgi?url=http://www.accessmedicine.com/lange.aspx
2. A website showing normal histology slides
http://vslides.unsw.edu.au/
3. On student computers: Go to Class Menu‐School of Medical Sciences, UNSW, Anatomy, Neurohistology
pdf file
4. On student computers: Digital Atlas of Electron Microscopy

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Practical 12: Internal Capsule and Horizontal Slices of the Forebrain
Specific objectives
1. To identify the components of the forebrain in horizontal sections.
2. To identify the caudate nucleus, globus pallidus, putamen and the relationship of these grey masses to the
thalamus and cerebral cortex.
3. To identify the internal capsule and predict the results of damage to it from a knowledge of its chief fibre
components.
4. To identify parts of the ventricular system and their relationship to the basal ganglia, thalamus and limbic
structures.
Learning activities
1. Examine brainstem specimens, and identify the thalamus and third ventricle just anterior to the
midbrain. The cavity of the lateral ventricle lies dorsal to the thalamus with the caudate nucleus in its
lateral wall. Identify the head, body and tail of the caudate nucleus. Identify the fibres of the trumpet‐
shaped internal capsule lateral to the caudate nucleus and thalamus. The internal capsule carries fibres
(in both directions) between the cerebral cortex and subcortical structures. Many of its fibres continue
inferiorly in the base of the cerebral peduncle (and appear to converge onto it). The concavity on the
lateral side of the internal capsule is occupied by the lentiform nucleus, which made up the putamen
laterally and the globus pallidus in its deep medial part. Identify the internal capsule on prosections of
the forebrain white matter. Identify the fibres of the optic radiation passing backwards from the
internal capsule to the visual cortex in the occipital lobe. (Photos of good dissections of the internal
capsule and white matter are also available in BrainStorm).

2. The horizontal brain slices give a good insight into the internal organisation of the forebrain provided
that they are kept in order and are handled with care. Using the slices identify the structures described
starting from the medullary centre of the hemisphere ‐ the large mass of white matter which is above
the lateral ventricle. This region contains fibres which run in different directions: many are commissural
fibres from the corpus callosum, others run towards the cortex from the direction of the thalamus
(forming the corona radiata). The commissural fibres of the corpus callosum form the roof of the lateral
ventricle. The dorsal part of the body of the lateral ventricle may actually appear in the central part of
the second section.

3. In the next sections identify the head and tail of the caudate nucleus, the lentiform nucleus, the
thalamus and their relationship to the internal capsule. Identify the two parts of the lentiform nucleus:
the globus pallidus and the putamen. Lateral to the putamen are the external capsule, claustrum and
the insular cortex. In several sections the caudate nucleus appears twice (its head and tail) as it circles
around the lentiform nucleus. The caudate nucleus runs in the lateral wall of the lateral ventricle.
Identify the anterior horn, body, collateral trigone, posterior and inferior horns of the lateral ventricle.
Notice that most of the lateral ventricle surrounds the thalamus, while its inferior horn extends into the
temporal lobe and the posterior horn into the occipital lobe. Look into the inferior horn and identify in it
the large hippocampus, covered by a sheet of white matter called the alveus. In the body, collateral
trigone and inferior horn you may identify the choroid plexus; however this part of the specimen is only
loosely attached to other structures and it may be missing. If present, notice that the attachment of the
plexus is along a line next to the fornix (see below).

4. Identify the fornix. This is a major tract of the brain which runs virtually free for most of its length. The
majority of its fibres arise from the hippocampus and appear on its medial edge in the form of the
fimbria which is the tail of the fornix. Then the fornix forms the medial wall of the lateral ventricle until
it passes in front of the interventricular foramen to enter the gray matter of the hypothalamus. The
fornices of the two hemispheres are, above the third ventricle, attached to each other and many fibres
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pass from one to the other: this is the hippocampal commissure. The dorsal surface of this commissure
is attached to the undersurface of the corpus callosum. In front of the interventricular foramen is the
column of the fornix which then passes through the hypothalamus to terminate in the mammillary
body.

5. Study the internal capsule in the slices. Describe the position of its 3 major parts: anterior limb, genu
and posterior limb. Note the three parts of the posterior limb: lenticulothalamic, retrolenticular and
sublenticular. Discuss the position of the fibre systems that run through these parts Draw a horizontal
section of the internal capsule and indicate on this drawing the location of the following fibre tracts
travelling in it: pyramidal tract (corticospinal + corticobulbar tracts), parts of the corticopontine
system, anterior, middle and posterior thalamic radiations, including the acoustic and radiations.
Discuss the clinical significance of the internal capsule. Predict the effect of a vascular lesion involving
the posterior limb (lenticulothalamic part)?

6. Study the white matter passing through the corpus callosum. Identify the major parts of the corpus
callosum ‐ the genu, body and splenium. The fibres which pass through the genu form the medial wall
of the anterior horn, and connect the frontal poles to each other.

7. Identify the anterior commissure. The cut fibres of this major commissure appear on the medial surface
of the hemisphere just anterior to the columns of the fornices. A fortuitous section may show the more
lateral parts of this fibre bundle, passing through the basal ganglia towards the temporal lobes, which
the anterior commissure connects.

8. Examine CT and MRI scans in the axial (horizontal) plane. What differences can you note in the
appearance of the major structures within the skull (brain (grey matter and white matter), ventricles,
paranasal sinuses, skull itself) in the two types of image? Try to identify as many as possible of the
features listed in learning activities above in these images.

9. Full sets of labelled MRI and myelin stained cross‐sections are available in BrainStorm and can be
accessed from the Cross‐sections menu. In your own time review your knowledge by going through the
Horizontal Forebrain and Horizontal MRI sections in BrainStorm and identifying the structures listed
above. You can toggle between myelin‐stained and MRI images by using the MRI/Tissue button.

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Practical 13: Motor Systems Physiology: Reflexes
Principal teacher: A/Prof Paul Bertrand
Introduction:
The motor system is the set of neurons, organised in feed‐forward and feedback networks, which is primarily
involved in the production of movement through contraction of muscle. One method to study the control of
muscle contraction is to record the depolarization of muscle in response to transmitter release at the
neuromuscular junction. Such a recording, known as an EMG or electro‐myogram, does not have a simple
relation to the force or velocity of muscle contraction, but rather reflects the electrical activity of the many
muscle fibres that make up an individual muscle.

In this science practical class you will record EMG potentials from the elbow flexors and extensors during a
variety of functional tasks and movements. After each experiment there are questions for you to answer as a
group. These questions will prompt you to think about what the experiment lets you infer about how the
nervous system controls movement.
Aim of the Learning Activity
1) You should understand the basic principles of EMG operation in order to appreciate research results
and clinical techniques that utilize these recordings.
2) You should appreciate how these EMG measurements enable you to draw conclusions about motor
control mechanisms such as:
• agonist/antagonist pairing
• the role of feedback in maintaining muscle position
• the feed‐forward nature of ballistic movements.
Equipment Required:
• Disposable Adhesive Electrodes (15 per station)
• Gauze pads for abrasion; Alcohol swabs
• Micropore tape
• PowerLab station; Button‐press trigger device; BioAmp leads
• Cloth strap
• 1 kg weight

Experiment 1 – Setting up
• Ask any volunteers to read the Participant Information Statement and then sign the consent form if they
choose to continue as a subject.
• Prepare electrode sites over Biceps Brachii and Triceps Brachii by lightly abrading skin over the muscle
belly using a gauze pad, and then thoroughly cleaning the area with an alcohol swab.
• Attach the disposable adhesive electrodes to the BioAmp leads and attach leads to the electrodes.
Channel 1 should be Biceps, Channel 2 Triceps.
• Wait until skin is dry and then fix electrodes to the skin. The positioning for the biceps and triceps
electrodes are shown in Figure 1.
• Ensure that the wires are neatly arranged and taped securely.
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Figure 1: placement of biceps and triceps electrodes
• Make sure your PowerLab is switched on (at the back) and you are logged into the computer to which
the PowerLab is connected. The PowerLab must be on before you start LabChart7.
• On the desktop, double click on the " LabChart7" icon. This opens the PowerLab module which
records your data. You then need to open an appropriate settings file: at the welcome screen choose
"Open" and use the dropdown menu to select: pp_class on 'Adunsw\data\medicine"
• Click into the directory ‐> PowerLab/Chart Settings/EMG.adiset
• Press the on‐screen button labelled "Start" to begin collecting data. The experimenter should tap the
EMG leads with their fingers to determine if the system is picking‐up a response from the electrodes.

Question: What happens to the EMG signal when the electrodes are tapped? Could this be a problem
during EMG recordings? What could we do to avoid this?

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Experiment 2 – Neural control of the force of contraction
• Continue using the settings file: EMG.adiset
• Seat the subject comfortably and ask them to grip the bench, and then to exert a weak elbow flexion.
Then have the subject execute a strong elbow flexion.
• Repeat this procedure for elbow extension.
• Examine your data: use the scroll buttons if required to show parts of the trace that have scrolled out of
sight. You may find it useful to adjust the vertical axis by dragging the numbers so that the signal occupies
about a half to two thirds of the vertical axis, or change the horizontal compression. Ask your
demonstrator for help if you need it.
• Stop the recording, then move the Waveform Cursor along the EMG trace until you find the peak reading
during a weak contraction. Read off the amplitude from the Range/Amplitude display at right and write
this number in your table.

Contraction muscle Peak EMG (Volts) Apparent frequency (Hz)
weak flexion biceps
triceps
strong flexion biceps
triceps
weak extension biceps
triceps
strong extension biceps
triceps

Question: What happens to the size and frequency of the EMG signal with increasing force of contraction?
Why?
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Experiment 3 – Joint movement depends on the activity of all muscles that act on it
• Continue using the settings file: EMG.adiset
• Ask the subject to hold a weight in their palm. Approximately 1 kg is a good weight.
• Ask the subject to close their eyes while holding the weight without generating any arm tension greater
than that required to hold the weight. At an unexpected moment, apply a brief tug to the strap at the wrist
to cause an elbow extension.
• Record your observations on movement at the elbow and peak EMG measurement in the table below. You
can have the subject rest their elbow on the bench if you are finding it difficult to separate shoulder and
elbow movement.
• Now ask the subject to produce EMG activity in both Biceps and Triceps with their elbow bent at 90
degrees and palm up so that the EMG in the biceps is approximately the same size as it was while holding
the weight. This is known as co‐contraction of antagonist muscles, and tends to stiffen the joint.
• Ask the subject to close their eyes but maintain the steady co‐contraction. At an unexpected moment,
apply a brief tug to the strap at the wrist to cause an elbow extension.
• Note the EMG response and the size of the resultant movement at the elbow in the table below.

Experiment muscle Peak EMG Elbow movement

holding weight biceps
triceps
co‐activation biceps
triceps

Question: Compare the size of resultant movement and EMG changes after the tug to the co‐contraction
situation. What is different in these two situations?

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Experiment 4 – Reflex control of posture using feedback
• From the file menu open the settings file: EMG_trigger.adiset.
• Now when you click “Start recording”, the computer will wait until the red button is pressed and then
released before signals will be captured. Data will be captured from 100 ms before the button was
released until 400 ms after the button was released.
• We will test the subject’s reaction time as a measure of voluntary activity. We will then compare this with
the time for a reflex postural correction. You will use the button‐press trigger device in this exercise (large
red button connected to PowerLab).
• Click Start and hold the red button down.
• Ask the subject to hold their elbow at 90 degrees until they hear the button being released. Ask them to
produce a short elbow flexion movement as quickly as possible after they hear the button sound.
• Record the time it takes to produce biceps EMG voluntarily after the stimulus. Repeat this five times and
take the average.
• Now have the subject to hold the button‐press trigger device and press the button against the underside of
the bench using a moderate elbow flexion force.
• Ask them to close their eyes, and then apply a brief tug to the strap so that the elbow is extended making
the button pull away from underneath the bench causing it to be released.
• Record the time it takes to produce the first peak in biceps after the stimulus (button release). Again
repeat five times and take the average.

Experiment biceps response time average of 5 response times
voluntary activation
(reaction time)
activation by wrist pull

Question: Do you think the biceps response you recorded in the second case is voluntary activity? Why?

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Experiment 5 – Ballistic movements using feed‐forward activity
• Go to the file menu and return to the settings file: EMG.adiset
• Ask the subject to sit in a low chair and hold the red button protruding from the bottom of their fist. They
should press the red button against the SIDE of the bench by applying a small elbow extension force. The
shoulder joint should be abducted to approximately 90 degrees, and the elbow joint extended to
approximately 145 degrees.
• Determine the arc taken by the subject’s finger when they produce a pure elbow flexion movement from
the starting position. Have an experimenter place their finger as a target along this arc so that the subject
has to make an elbow flexion of approximately 45‐60 degrees to reach the target.
• Ask the subject to make an elbow flexion movement, as fast as possible, from the start position with the
button pressed down to the target.
• Observe the Biceps and Triceps EMG activity. Repeat a number of times to see the most common patterns.
Draw your response here, indicating timing.

Questions:
• When does the Biceps EMG start relative to the button press? Is this what you would expect?

• What happens to the Triceps EMG and when? How do you explain this?

• Do you see a second burst of Biceps EMG on any trials? Why might this be useful?

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• Repeat the procedure with subject’s eyes closed by getting them to move to a standard position as fast as
possible. Then, without causing any increase in tension in the strap and thereby warning the subject,
attempt to prevent the elbow flexion movement by holding the strap. Draw your response here, indicating
timing.

Questions:
• How does the pattern of Biceps and Triceps EMG in this restricted movement compare with the full
movement? Is this what you would expect?

• What does this tell you about the way in which the nervous system controls rapid goal directed
movements?

• Compare the response you record when the subject is warned that their movement will be restricted with
the response obtained without warning. What is different in these two situations?

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Practical 14: Human aspects of living with neuro‐degenerative disease
Principal teacher: Dr. Úte Vollmer‐Conna

Material will be provided as required at Prac sessions.
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Practical 15: Coronal Slices of the Forebrain
Specific objectives
1. To identify the components of the forebrain in coronal sections.
2. To identify the specific rostro‐caudal order of the main internal structures of the hemisphere and their
relationship to the surface features of the cortex.
3. To identify the main components of the limbic system in gross dissections, coronal slices and MRI’s.
4. To identify parts of the ventricular system in coronal sections and MRI’s and observe their relationship
to the basal ganglia, thalamus and limbic structures.
5. To identify the major arteries on the base of the brain.
Learning activities
1. Divide each set of the coronal sections into three groups and treat them in sequence as sections of the
anterior, middle and posterior thirds of the hemisphere. The middle third extends from the olfactory
trigone to the splenium of the corpus callosum and is the most complex region. It is advisable to
compare the coronal sections with the intact hemispheres in order to determine the exact position of
each section, and also to correlate them with the horizontal slices to gain a fuller understanding of the
three dimensional organization of the hemisphere.

2. In the first set of sections identify the genu of the corpus callosum and the anterior horn of the lateral
ventricle. Identify the head of the caudate nucleus and note that it merges basally with the putamen.
The region where these nuclei merge is called the nucleus accumbens. Dorsally, the head of the
caudate nucleus and putamen move apart but they are still connected to each other by strands of gray
matter passing through the anterior limb of the internal capsule. This gives the complex a striated
appearance, hence the name corpus striatum. Medial to the nucleus accumbens, in a ventral
continuation of the septum pellucidum, is the septal area which is an important part of the limbic
system. It is at this level that the anterior commissure comes into section (check again the medial edge
of the sections), together with the optic chiasm. Behind the genu and below the body of the corpus
callosum, the septum pellucidum separates the anterior horns of the lateral ventricles from each other,
while more posteriorly the septum becomes gradually smaller and the ventricles move away from the
midline.

3. In the sections of the middle third, observe the internal organization of the lentiform nucleus, with the
two segments of the globus pallidus medially and the darker putamen laterally. Note in the sections
that the putamen extends beyond the globus pallidus in all directions. Medial to the lentiform nucleus
identify the internal capsule. Follow this white band of fibres posteriorly into the crus cerebri.

4. Identify the major masses of the diencephalon. Identify the thalamus, on the medial side of the
posterior limb of the internal capsule and on its ventral surface, the optic tract. The grey matter around
the basal part of the third ventricle is the hypothalamus, which is delimited posterolaterally by the
cerebral peduncles.

5. Ventral to the thalamus and lateral to the hypothalamus is the subthalamic region. Identify the
subthalamic nucleus which appears just dorsal to the fibres of the internal capsule. In the caudal
sections through the thalamus you may identify parts of the midbrain such as the substantia nigra and
red nucleus.

6. Focus your attention on the temporal lobe. In the more anterior sections, identify the amygdaloid
nucleus, large limbic nucleus which merges with the overlying cerebral cortex (the uncus). Immediately
behind the amygdaloid nucleus the inferior horn of the lateral ventricle starts. The inferior horn
contains the hippocampus in its floor and the tail of the caudate nucleus in its roof. The medial wall is
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closed off from the subarachnoid space by the choroid plexus, a pial‐vascular structure, which may or
may not still be present in your sections.

7. In the posterior set of sections identify the collateral trigone of the lateral ventricle: this is the part
connecting the body of the ventricle with the posterior and inferior horns. Lateral to the collateral
trigone and posterior horn a distinct white band, the optic radiation can be observed.

8. Now examine MRI sections in the coronal plane and try to identify as many as possible of the structures
listed above. These are available in the lab (film) or in BrainStorm (Coronal MRI submenu). In your own
time also examine the radiographic appearance of the abnormal brain by viewing the pathology images
in BrainStorm (Histology/Pathology submenu).

9. On whole brains examine the major arteries that supply the forebrain. First identify the internal carotid
artery and its terminal branches, the middle and anterior cerebral arteries. On each side the internal
carotid artery is joined to the posterior cerebral artery (a branch of the basilar artery) by the posterior
communicating artery. The anterior cerebral arteries are joined by the anterior communicating artery,
completing an anastomotic ring called the Circle of Willis, or the cerebral arterial circle. (Theory relating
to blood supply will be covered in a lecture.)

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Practical 16: Visual Physiology
Principal teacher: Dr. Richard Vickery
Introduction:
Visual function relies on the optics of the eye to form a clear image on the retina. Here the neural circuitry of
the retina turns this image into a pattern of neural activity, beginning with photoreceptors, and ultimately
exiting in the optic nerve as axons of retinal ganglion cells. In this practical class we will examine both the optics
of the eye, and the neural organisation that gives rise to vision.
Aims:
1. Gain an understanding of how to measure myopia and hyperopia and how to correct for them.
2. Be able to describe and distinguish between optical and neural limitations on human vision.
3. Be able to describe colours and colour vision on the basis of the three classes of cone photoreceptors.
4. Describe the physiological limitations on depth perception, and describe how to assess stereoscopic vision.

1. Pupillary light reflexes
Test the pupillary light reflex. Shine a light into one eye, and watch carefully: you should be able to see the
pupil constrict.

Allow a minute for the subject to recover.
Test the consensual pupillary reflex. Shine the light into one eye and watch the pupil of the other eye.

Why do you think the nervous system has this reflex?

Which reflex is more likely to be affected by brain damage?

2. Use the ophthalmoscope to examine the retina.
The pupil of the eye only looks black because not much light is reflecting back from it. The ophthalmoscope
allows you to shine a beam of light into the eye and look into the pupil "behind" the light beam.

For good visualisation of the retina a mydriatic eye drop is usually given to dilate the pupil. In this class we will
examine the retina without using a mydriatic. With luck or practice you will be able to see some blood vessels
on the retina, the optic disc (the site at which the optic nerve and vessels leave the eye), and maybe the fovea
(the area which is specialized for detailed vision).

• The on‐off switch for the ophthalmoscope is the ring on the collar.
• Test where the spot of light shines.
• You shine the light into your subject's pupil while looking through the ophthalmoscope from the opposite
side.
• You will need to be close (15‐20 cm) to your subject's eye.
• Initially set the dial to “0”; if you can see the retina but it is blurry, then turn the ring to correct for the
optics of you and your subject.
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• The light might make the subject's eye water, but they should try to keep their eye still. You will need to
move your head behind the ophthalmoscope to see more of the retina through the constricted pupil.
• Find a blood vessel. Make the image as sharp as possible by turning the ring.
• Now try and follow the vessel to the optic disc (a white spot with all the blood vessels radiating out)

Draw what you can see here.

3. The blind spot
All of us are blind ‐ at least in part of the visual field! This blind spot represents the area where the optic nerve
passes through the retina (the optic disc) and so there are no photoreceptors there. The brain "fills in" the hole
in our visual field with the information from the surrounding rods and cones and from the other eye if it is
open.

Map your blind spot on this sheet of paper.
• Close your left eye and fixate on (stare at) the cross.
• Starting about 10 cm from the page, move your head slowly backwards away from the page.
• When the dot disappears, make it bigger by colouring around it with a pen and then try again.
• By repeating this procedure, you will be able to map the position, shape, and size of your blind spot. The
area you have coloured in reflects the optic disc, relative to the point of fixation (the cross) which falls on
your fovea.
+ •
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4. Visual acuity: the Snellen Chart
Test whether you have 20/20 vision (now called 6/6 vision due to the change from feet to metres).
• Stand 6m from the chart on the tape mark on the floor.
• Cover one eye ‐ the chart is designed to test monocular acuity.
• Have a lab partner point to a row of letters for you to read out.
• If you get them all correct, advance to the next smaller row of letters.
• With normal vision you should be able to read the line of letters labelled 6m ‐ this corresponds to reading
the 20 foot set at the 20 foot mark ‐ hence 20/20. If you can read the lowest line (5m), then your vision is
better than average (6/5). The number next to each line indicates the distance at which a person with
normal eyesight can read the letters.
• To experience the blurry world of a myopic person, now try and measure your acuity looking through a
convex lens (magnifying glass).
Write down your normal visual acuity. Write down your acuity through the convex lens.

Try and draw the letter A in the 2 x 2 box just by colouring squares ‐ can you do it?

Now try and draw the letter A in the 5 x 5 box just by colouring squares ‐ can you do it?

Now translate your acuity, to the size of the letter image formed on your retina.
The focal length of the eye is 17mm. You are standing 6 m from the chart. This means the size of the image on
your retina will be the height of the letters in the smallest line you could resolve multiplied by (17 / 6 000).
Measure the height of the letter and do the calculation.

Approximate size of image of the letter on the retina is: (don't forget your units).

5. Visual acuity: accommodative power of the lens
The power of a lens is measured in dioptres. The lens in the human eye is capable of changing its power, which
permits us to focus up close or far away. You will measure your accommodative power and confirm the power
of the lens in any spectacles you wear.
• Determine your near point. Gradually bring this page of text up to one eye along the metre ruler and
determine the distance at which it first becomes blurry. Have someone measure this distance and write it
down in metres.
• If you are myopic (short‐sighted) determine your far point, which is the furthest distance that you can see
objects sharply. Do this by having someone move this page of text away from one eye along the metre
ruler, and determine the distance at which it first becomes blurry. Have someone measure this distance
and write it down in metres. If you do not have myopia, this distance should be infinity.
• Calculate your accommodative power = 1/(near point) – 1/(far point)
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• If you wear glasses, determine their power by measuring your near point with and without glasses. The
power of your lenses = 1/(near point with glasses) – 1/(near point without glasses)
Lenses to correct myopia will have a negative power (diverging or concave lens).
• If you don't wear glasses, you can use the same technique to test the power of the convex lens that you
used to simulate myopic blur in exercise 4.

What is your accommodative power? is this normal for someone of your age?

What is the calculated power of the lens? If the lens is your glasses, does this power match the optometrist's
prescription?

6. Colour vision studied using colour after‐images
Switch on the computer. Press the "Start" button at bottom left of the Windows screen, and then choose
"Class Programs". On this screen choose the button "Other Physiology" and the option from there "Sensory
Physiology: Colour After‐effect". Follow the instructions on the screen.

Do the after‐images let you determine that there are separate colour channels?

Would there be any difference to the colour wheel and the primary colours for people with only 2 functional
cone types?

Would there be any difference to the colour wheel and the primary colours for a species with 4 cone types?
Would they see more colours than us?

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7. The Ishihara test for colour blindness.
This test consists of a series of colour plates with numbers or simple winding paths formed from coloured dots.
Subjects with colour blindness will perceive a different number or path.

• Put the Ishihara book in a well‐lit position where everyone in the group can see it clearly.
• DO NOT TOUCH THE IMAGES ON THE PAGES AS YOU WILL CAUSE DISCOLOURATION WHICH WILL
INVALIDATE THE TEST FOR FUTURE GROUPS.
• Start at the beginning and all agree on what you see. Some pages will show a number, some will have a
path to be traced from one "X" to another "X", and some pages will be blank.
• If any member of your group sees a different number or path from the rest of the group, you can obtain a
copy of the test answer booklet and carefully work through the plates with him, to determine the form of
colour blindness that he has (colour‐blindness is sex‐linked and affects about 7% of males and <1% of
women).

8. Clinical assessment of stereopsis
This simple clinical test of stereopsis called the Stereotest relies on polarising glasses and a gel film with two
layers that transmit differently polarise light. This test provides an easily administered check of stereoscopic
depth perception. Its purpose is to measure how well the two eyes can discern differences in the distances of
objects from the observer. Determinants of depth such as different object size, overlapping of objects, and
perspective have been excluded from the images.

• To administer the tests, hold the picture straight in front of the observer, to maintain the proper axis of
polarization.
• Avoid reflections from the shiny surface.
• The graded tests are standardized for 40 cm viewing distance, but minor variations have little effect on the
score.
• The polarized viewers must be worn when viewing the pictures (over the subject's glasses if they normally
wear them).
• The three tests are each used under different circumstances:
1) The House Fly establishes the presence of gross stereopsis, and is especially useful in testing
children. Have the observer try to "pinch" the wing of the fly between thumb and forefinger
(provides a guide to the extent of stereopsis).
2) The Circle patterns provide a finely graded series which tests fine depth discrimination. Within
each square are four circles. Only one of the circles should appear forward of the plane of
reference for those having normal fusion. (The angles of stereopsis (seconds of arc) at 16 inches
subtended by each test circle are: 1 (800 sec), 2 (400 sec), 3 (200 sec), 4 (140 sec), 5 (100 sec), 6
(80 sec), 7 (60 sec), 8 (50 sec) and 9 (40 sec).
3) The series of animals facilitates the testing of young children. In each line one of the five animals
appears forward of the others.

What is your stereoacuity?

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9. Magic Eye Pictures
Magic Eye pictures or Single Image Stereograms as they are more generically known, present a stereoscopic
perception of depth without the need for any specialised viewing apparatus. Inability to see Single Image
Stereograms may relate to problems with stereopsis or with ocular muscle control of gaze. Provided that you
have some stereoscopic depth perception (at least 100 seconds of arc on the previous test), then you should be
able to see these Single Image Stereograms. Stable gaze control is required because to view the Single Image
Stereograms the subject must uncouple their vergence and accommodation. For this reason, Magic Eye
pictures are often used to train children in the control of their extra‐ocular muscles in treating conditions such
as mild strabismus.

View some examples of Single Image Stereograms here:
http://pionet.net/~k0brd/stereo/sirds/index2.html
http://www.cg.tuwien.ac.at/~mroz/sirds/history.html

How is the brain able to reconstruct depth from a flat Magic Eye picture? Why must accommodation and
vergence be uncoupled?

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Practical 17: CNS Pharmacology
Principal teacher: Dr. Nicole Jones
Aim
To observe and evaluate behavioural responses of animals to various pharmacological agents affecting the
central nervous system.
Introduction
A behavioural screening test is usually applied to new compounds to determine preliminary information on
their activities and toxicities, and provide clues for their classification. Most screening tests are performed on
animals, usually mice and rats, since it would be unethical to test drugs with potentially adverse side‐effects on
human beings. After drug administration, the animals are carefully monitored for various parameters, such as
awareness, mood, motor activity, central nervous system excitation, muscle tone, reflexes etc., and scored on a
numerical scale. From the scores given to the observations, tentative conclusions can be made about the
pharmacology of the compound.
Behavioural screening studies are important in ensuring that pharmacologists are aware of the distinctive
effects, efficacies and toxicities of the drugs. However, such studies require large quantities of animals, which
cause financial and animal ethical concerns. Hence, the development of alternative approaches for teaching
and researching behavioural pharmacology has become a worldwide issue.
In this practical you will be introduced to an alternative approach to teaching behavioural pharmacology,
involving the use of pre‐recorded video to demonstrate drug screening procedures in live animals. With the use
of video sequences within the program, together with tutorial style questions, you will have a store of visual
information available regarding the appropriate behavioural effects of some drugs, such as CNS stimulants,
sedatives and narcotic analgesics.
Methods
PART 1. Observation of behaviour responses to pharmacological agents
Students will observe computer‐based demonstrations and recordings of the actions of various CNS active
agents and the responses of animals to these agents, and will be asked to comment upon and evaluate these
during the class.

Make sure you read the definitions of various behaviours that may be observed in the tests before the class
starts. Also make sure that you attempt the questions listed below at the end of each section.
Use the multimedia CD‐ROM to observe the behavioural effects of the following agents:
a) Hypnotics/Sedatives: Barbiturates//Benzodiazepines
b) Opioids: Morphine
c) Stimulants: Amphetamine/Cocaine/Picrotoxin

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Definitions of behaviours

Ataxia ‐ loss of the ability to coordinate muscular movement.
Clonic convulsion ‐ uncontrollable contractions of muscles marked by alternating contraction and relaxation
of the muscles.
Corneal reflex ‐ reaction of the eye to changes in light (change in the size of the pupil)
Dyspnoea ‐ difficult or laboured breathing.
Hypertonia ‐ excessive tone of the skeletal muscle.
Hypotonia ‐diminished tone of skeletal muscle.
Miosis ‐ contraction of the pupil.
Mydriasis ‐ dilation of the pupil.
Opisthotonus ‐ a form of spasm consisting of extreme hypertension of the body.
Piloerection ‐ erection of hair.
Ptosis ‐ drooping or closure of the eyelids.
Salivation ‐ secretion of clear alkaline from mouth.
Sedation ‐ defined as the act or process of calm.
Spasms ‐ sudden violent involuntary contraction of muscle.
Stereotypies – persistent repetition of stereotyped behavior, eg, preening and sniffing the floor.
Straub tail ‐ raising the tail in the air.
Wet‐dog shakes ‐ twisting & shaking of the head and neck.
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Use the following table to indicate the type(s) of behaviours you observe for each of the agents tested.

Behaviour Hexobarbital Morphine Amphetamine Picrotoxin
Ataxia
Corneal reflex
Clonic convulsion
Dyspnoea
Hypertonia
Hypotonia
Miosis
Opisthotonus
Piloerection
Ptosis
Respiratory depression
Salivation
Sedation
Spasms
Stereotypies
Straub tail
Wet dog shaking
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PART 2. ELEVATED PLUS MAZE
The elevated plus maze is a common behavioural test used to assess fear and anxiety in rats and mice. The
equipment used for this test is an elevated 4 armed maze, 2 of the arms are completely open, while the other 2
arms have enclosed / raised sides. This test is used to determine whether drugs / treatments have potential
anxiolytic (reduce anxiety) or anxiogenic (increase anxiety) actions.

Students will observe the behaviour of two rats on the elevated plus maze. Each rat has been injected with a
drug and has been placed in the centre of the elevated plus maze. Each video file will contain 10 min video
recordings from 2 individual rats. You will receive a short demonstration of how to measure the behaviour
using the elevated plus maze.

For each rat your group will need to note down and measure:

a) Rat ID
b) Total time spent in the open arms (using timer)
c) The number of entries into the open or the closed arm of the maze.
Note: each entry is counted as when the whole of the rat’s body has entered an arm of the maze.

You should express your results according to the table below. Once you have filled in the table provided – add
your results to the class data

Rat ID # entries # entries closed # entries % open entries Time spent in
open total / Total open arms (sec)

When all of the class data has been collected and the codes for the treatment groups have been revealed:

• Calculate the mean and standard deviation (% open entries) for both control and diazepam treated
groups:
1. Open GraphPad Prism via the following path: Class programs \ Physiology and Pharmacology \
Utilities and Office applications \ Graph Pad Prism
2. Select “start with an empty data table”, Choose Graph “Column bar graph, vertical”, choose to plot
“mean with SD”
3. Enter control values (% open entries) in column “A” and diazepam values (% open entries) in column
“B”
4. Click on the “analyse” button, Under “column analyses, select t‐test (and non‐parametric tests)
5. Select “paired test”, two‐tailed, and use 95% confidence intervals
6. Click “OK” and your Prism will perform the t‐test
7. Click on “Graph” to view your bar graph
8. Label each axis. “X” = Treatment, “Y” = % open entries

• Calculate the mean and standard deviation (Time spent in open arms) for both control and diazepam
treated groups.
Repeat steps 1‐8 (above), but use class data for Time Spent in open arms and label your “Y” axis on the
graph accordingly.

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Quiz for Part 1
a) Hypnotics/Sedatives
Q1. The barbiturates are known to produce exciting ataxia and hypotonia prior to anaesthesia. What is the
other predominant behavioural characteristic observed?
a. Opisthotonus
b. Clonic convulsions
c. Salivation
d. Mydriasis
e. Respiratory depression
Q2. A subject is given an unknown drug which produces anaesthesia. Upon awakening, the patient
suffers amnesia for 3 hours. The drug is likely to be a type of
a. Barbiturate
b. Opioid
c. Benzodiazepine
d. Cholinergic
e. Ether
Q3. Which of the following are the effects of benzodiazepines?
a. Reduction of anxiety and aggression
b. Anticonvulsant effect
c. Acts allosterically to increase of GABA for the GABAA receptor binding
d. Rapid eye movement (REM) sleep suppression
e. All o
b) Opioids
Q4. The distinguished behavioural characteristics of morphine as demonstrated in the video sequence
above include
a. Sporadic activity
b. Straub tail
c. Hunched posture
d. Walking on tiptoe
e. All of the above
Q5. In addition to the decreased pain perception and sedative effects, morphine also has effects on the
eye and typically produces:
a. Miosis
b. Ptosis
c. Mydriasis
d. Ptosis and Mydriasis
e. Ptosis and Miosis
Q6. Morphine produces its effects through activation of specific opioid receptors in the brain. The receptor
responsible for producing most of the analgesic effects is
a. δ receptor
b. μ receptor
c. κ receptor
d. σ receptor
e. α receptor
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Q7. The most prominent clinically useful effect of opioids is reducing pain. Which of the following are
unwanted effects of morphine?
a. Euphoria and respiratory depression
b. Sedation and dependence
c. Constipation
d. Both a and b
e. a, b and c
c) Stimulants
Q8. The major behavioral characteristic of amphetamine as demonstrated in the video sequence above is
a. Sporadic activity
b. Piloerection
c. Normal movement pattern
d. Stereotypies
e. None of the above
Q9. Amphetamine and cocaine are both drugs of dependence which are subject to abuse in the
community. They have very limited clinical use. The main use of amphetamines is in the treatment
of
a. Obesity
b. Attention deficit/hyperactivity disorder (ADHD) in children
c. Narcolepsy
d. Both a and c
e. Both b and c
Q10. The mechanism underlying the psychostimulant effects of cocaine is
a. Stimulation of catecholamine uptake
b. Inhibition of catecholamine uptake
c. Release of catecholamine
d. Non‐competitive antagonist of 5‐HT receptor
Q11. The convulsants form a diverse group of drugs which have varied mechanisms of action, but share a
number of important behavioural characteristics, including
a. Clonic convulsion, salivation
b. Spasm, salivation
c. Clonic convulsion, hyperreflexia
d. Spasm, wet‐dog shakes
e. Clonic convulsion, spasm
Q12. Picrotoxin acts as a
a. GABAA receptor agonist
b. GABAA receptor antagonist
c. Glycine receptor antagonist
d. Adenosine receptor antagonist
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Questions for Part 2

1. Are the means of control and diazepam treated groups significantly different?

2. How does diazepam work?

3. Why are we calculating open entries as a percent of the total number of entries?

4. Why is important to be blind to the treatment groups when studying animal behaviour?

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Page 79

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Station Instructions and Notes Questions

Safety Summary: Physiology Practical

A. Safety controls to be observed:

B. In the event of an emergency evacuation:

Signature: ........................................ Date: .........................................

iii) Calculate the theoretical equilibrium potential for both K+ (EK+) and Cl‐ (ECl‐) at each concentration gradient

RT ⎛ PK : PCl .[ K + ] o + [Cl −]i ⎞

TOTAL STEROID (nM)

Experiment muscle Peak EMG Elbow movement

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