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

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.


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.


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

11. CNS: Normal and abnormal Mr Patrick de Permentier / 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.


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 identify the following: body, costal demifacets, pedicle and lamina, articular, transverse and spinous processes. Identify the articular surface on the transverse processes for the tubercle of the corresponding rib.

Which vertebral joints are involved in joints between vertebrae? How do the parts of the thoracic vertebrae protect the spinal cord and spinal nerves?

2 Review the major features of a typical rib and identify: head with articular facets, neck, angle, shaft, and tubercle with its articular and non‐articular components. Use your lecture notes to consider how the head and tubercle of a rib articulate with the body and transverse process of the thoracic vertebra (costovertebral and costotransverse joints). Identify parts of the sternum: manubrium, body and xiphoid process.

What type of joint is found between the manubrium and body of the sternum? What is this point called? What structures lie within the costal groove?

3 On the articulated skeleton, observe the general shape of the thoracic cage (truncated cone, elliptical on cross section). Note 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.

Which costal cartilage articulates with the sternal angle?


Station Instructions and Notes Questions 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 layers of muscle (the external, internal and innermost 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.

What are the directions of muscle fibres in the three layers?

6 Identify the intercostal nerve and vessels (posterior intercostal artery and vein) that lie in the plane between the internal and 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).

Between which muscle layers do the neurovascular elements run?

II. HUMERUS and MUSCLES OF THE AXILLARY WALLS AND ARM Use the bones and wet specimens for the following stations. Station Instructions and Notes Questions

7 Use the isolated humerus and articulated skeleton to identify key bony landmarks at this bone’s upper end. Note the greater and 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).

What structures pass through the cervicoaxillary canal?

8 Use the wet specimens to identify pectoralis major, pectoralis minor, latissimus dorsi, subscapularis, teres major and serratus 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.

What are the functions of the muscles in bold to the left?

9 On favourable specimens you may be able to see branches of the brachial plexus to the muscles that bound the axilla (long thoracic nerve to the serratus anterior, pectoral nerves to the pectoralis muscles, subscapular nerve to the subscapularis muscle.

In what sort of surgery might these nerves be damaged?


III. THE LYMPHATIC SYSTEM AND AXILLARY LYMPH NODES Refer to your lecture notes concerning the general organisation of the lymphatic system. Station Instructions and Notes Questions10 Note that axillary lymph nodes have been removed from the

prosected specimens along with loose connective tissue, but you should be able to use your lecture notes to indicate to colleagues the expected positions of the five groups of axillary lymph nodes (central, apical, lateral or humeral, anterior or pectoral, and posterior or subscapular). Use your lecture notes to recall the pattern of lymph drainage between these groups.

Which lymph node group would first receive lymph from the arm? Which would first receive lymph from the breast? Why are lymph nodes enlarged when there is an infection or cancer in the area that they drain? How does palpation of enlarged lymph nodes help differentiate between infection and cancer?

IV. THE MAMMARY GLAND (THE BREAST) Station Instructions and Notes Questions11 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 should refer to your lecture notes and models while discussing the macroscopic structure of the breast with colleagues: • nipple, areola • mammary gland lobules, lactiferous ducts, lactiferous

sinuses • fibrous septa (suspensory ligaments)

How does the internal structure of the breast change with puberty, pregnancy/lactation and menopause?

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 eventually in the internal thoracic and axillary veins. There is some drainage into the posterior intercostal veins. The posterior intercostal veins are also connected (by the azygos system of 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).

What is the course that might be followed by metastases from the breast to the vertebral column and skull base?

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.


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 homozygous knock‐outs

Coschigano et al. (2000)

IGF‐R1 Insulin‐like growth factor type 1 receptor

Heterozygous mice live 26% longer than wild‐type

Holzenberger et al. (2003)

MSRA Methionine sulfoxide reductase A

Decreased lifespan and possible accelerated ageing in homozygous knock‐outs

Moskovitz et al. (2001)

p53 Tumour suppressor protein 53 Heterozygous mutant mice display signs of accelerated ageing

Tyner et al. (2002)

p66shc p66shc (SHC1) Roughly 30% increase in lifespan in ‐/‐ mice

Migliaccio et al. (1999)

PASG Proliferation associated SNF2‐like gene

Possible accelerated ageing phenotype due to disruption

Sun et al. (2004)

Pit1 Snell dwarf mouse Lifespan increase of 42% in homozygous mice

Flurkey et al. (2001)

Thdx1 Thioredoxin 35% increase in average longevity in transgenic mice

Mitsui et al. (2002)

XPD Xeroderma pigmentosum, group D

Possible accelerated ageing phenotype due to homozygous mutation

de Boer et al. (2002)

Human (Homo sapiens sapiens)

CKN1 Cockayne syndrome Type I Possible premature ageing due to recessive mutation

Henning et al. (1995)

WRN Werner syndrome Possible accelerated ageing due to recessive mutation

Yu et al. (1996)

Lamin A Hutchinson–Gilford syndrome Possible premature ageing due to dominant mutation

Eriksson et al. (2003)


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.


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.


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.

Sample Time

sampled (hrs) A620

Your data A620

Colleague’s data AverageA620

Cells/ml

0

2

6

8

10

12

14

18

Data for the other samples from your group:

Sample Time

sampled (hrs)

Cells/ml Sample Time

sampled (hrs) Cells/ml

0 0

2 2

6 6

8 8

10 10

12 12

14 14

18 18


Sample Time

sampled (hrs)

Cells/ml Sample Time

sampled (hrs) Cells/ml

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?


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


Fibroadenoma, breast

Ductal carcinoma, breast


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


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


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.


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?


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


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.


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.


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: .........................................


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


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.


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 in the outside bath

[KCl]o (mM)

Membrane Potential (mV)

Theoretical K+ equilibrium (Nernst)

Potential (mV)

Theoretical Cl‐ equilibrium (Nernst)

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?


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:

⎟⎟⎠

⎞⎜⎜⎝

⎛⋅=

i

om X

XzFRTV

][][ln

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:



2 3 4 5 6 7 8 9 2 3 4 5 6 7 8 9 2 3 4 5 6 7 8 91 1 1 1

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 150γ 0.958 0.946 0.927 0.902 0.858 0.817 0.750 0.770 0.753 0.740



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:

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), 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.

.: PClPK

Results and other notes

⎟⎟⎠

⎞⎜⎜⎝

⎛−++−++

⋅=oCliKPClPKiClKPClPK

FRTV o

m ][].[:][].[:

ln

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 to the nucleus. This practical involves investigating the binding of [3H]‐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. As a result, radio‐labelling (e.g. [3H]‐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.



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]

0 5 100.0

0.5

1.0

TotalSpecificNon-Specific

TOTAL STEROID (nM)

[BO

UN

D S

TER

OID

] (pm

ol)

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.

0

1

2

3

4

5

B/F

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.


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.


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 5192 10313 14794 25335 29766 47297 268 509 10910 19611 38512 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

← These are data obtained by the laboratory staff in setting

up the experiment.


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. Advanced students (i.e. 2nd 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


http://sfx.nun.unsw.edu.au:9003/sfx_local?issn=1533-6581&date=2000&volume=343&issue=21&spage=1506

http://sfx.nun.unsw.edu.au:9003/sfx_local?issn=1533-6581&date=2000&volume=343&issue=21&spage=1506

http://aac.asm.org/cgi/content/full/48/8/2787

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?


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?


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?


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?


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.


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


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.


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. .


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?


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?


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?


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?


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?


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


http://vslides.unsw.edu.au/VirtualSlideV2.nsf/id/8074FA

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?


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?


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?


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


http://info.library.unsw.edu.au/cgi-bin/local/access/access.cgi?url=http://www.accessmedicine.com/lange.aspx

http://info.library.unsw.edu.au/cgi-bin/local/access/access.cgi?url=http://www.accessmedicine.com/lange.aspx

http://vslides.unsw.edu.au/

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


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.


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.


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?


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?


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?


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?


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?


• 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?


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.


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


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.)


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.


• 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.

+ •


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)


• 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?


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?


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?


http://pionet.net/%7Ek0brd/stereo/sirds/index2.html

http://www.cg.tuwien.ac.at/%7Emroz/sirds/history.html

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


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.


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


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 open

# entries closed # entriestotal

% open entries / Total

Time spent in 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.


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


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 e. None of the above

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 e. None of the above



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?

aeb practical guide

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