Objectives
This course is not designed to be a general introduction to the whole of animal physiology. Instead, specific subjects have been chosen to illustrate the two major divisions of animal physiology; environmental and regulatory. Other subjects are covered by third year honours courses:
HBZ3Z01 Comparative Animal Physiology (osmoregulation and excretion);
HBZ3Z02 Experimental Zoology (muscle and metabolism);
HBZ3Z04 Ecological Animal Physiology (temperature and energetics).
Animal Physiology is a pre-requisite for each of these.

Syllabus. The approach will be comparative, with the joint themes of adaptation to the environment, and regulation and control:
Respiration in water and air, in insects, fish, amphibians, birds and mammals. Transport of oxygen and carbon dioxide by blood. Circulation. Resting and action potentials in excitable cells. Synaptic integration. Sensory receptor mechanisms and sense organs. Hormones and neuroendocrine control.

Recommended books. The best book which covers this course is:
Schmidt-Nielsen, K. Animal physiology: adaptation and environment. Cambridge Univ. Press.
The library has several copies of the 3rd edition (1983; QP 31.2 SCH), which is also available in the Book Centre (First Street / Silundika Avenue). The 4th edition (1990) is available in Kingstons on campus. There are only minor differences between the 3rd and 4th editions in the subjects covered in this course.
Another useful book, particularly for those students who will take Experimental Zoology, is:
Keynes, R D and Aidley, D J. (1981). Nerve and muscle. Cambridge Univ. Press. This is available in the Book Centre.

Other equipment. You will need to bring a lab coat, dissecting kit, drawing paper, pencil and sharpener, and calculator to the practicals: the calculator may also be needed in the examination.

Practicals have been designed to illustrate the theory course, to show the experimental method, and to give experience in analysing data and writing reports. Another aim, to show specific techniques, is harder to achieve with the large class size. Nevertheless, some useful methods are practised, such as chemical analysis, quantitative microscopy, dissection and drawing, and spectrophotometry. It is up to you to make the most of the opportunity.

One reason for handing out all practical schedules together at the start of the course is to enable you to read them before doing the practicals. It is most important that you do so - this will enable you to work much more effectively.

Most of the equipment and reagents will be found on your work benches. A few items, such as balances, are available centrally on the side bench. At the end of the practical class the benches should be left clean and as tidy as possible. Unused or intact animals should be returned to the technicians, who should also be informed of any breakages or non-functional equipment. Dirty glassware should be rinsed and placed on the draining board at the end of the bench, and dead animals and rubbish put into the bins. Check that all water and gas taps and mains switches are turned off.

Safety. For your own and the class' safety you should comply with the following regulations:
1. Wear a laboratory coat.
2. Be careful what you put into your mouth. Use a pipette bulb when pipetting strong acids, alkalis or poisons. If these are not available, ask for them.
3. There is a first aid cupboard on the laboratory wall. Ensure you know the location of fire extinguishers. There is a safety shower in the Part II Experimental Laboratory.
4. On no account may food be eaten in the laboratory; smoke outside if you must.

Equipment. Some of these practicals involve handling expensive equipment, such as spectrophotometers or microscopes. Make sure you know how to use this; if in any doubt, ask a demonstrator.

All practicals will be marked and will contribute to your CAP. Because of the large class size, many practicals will be performed in two groups, switching over between Monday and Thursday. Write-ups are to be handed in BEFORE the start of the next practical. No late submissions will be accepted. Repeat students must complete the 1995 practical course, which differs from that of 1994.

Practicals should be written up on lined A4 paper, white drawing paper, and graph paper. You will lose marks for poor presentation, for example on old computer paper. Write-ups should in general be in the form of a scientific paper. Points to include:
Abstract - A brief (half page) description of your findings and conclusions. Include the major numerical results.
Introduction - Background information (with specific references) and the particular aim of the experiment, in your own words.
Methods - Should include all modifications, and the reasons for these, but no need to reproduce the whole schedule.
Results - Your own raw data, and calculations on them, all explained in full. It is inadequate to only present the results sheet from the practical without explanation.
Discussion - Explanations of the patterns shown by the data, expected and unexpected. Faults with the experiment and how it could be improved. Comparison with published results. You should give a specific reference for each factual statement made.
References - List references consulted, in the standard form used in this handbook. Only include those which you have actually seen and referred to in the text of your write-up.

The write-up should be your own individual work, not a joint effort or copy of someone else's report. All such co-authored reports will get a mark of zero.

No essay will be set as this course immediately precedes the examinations. Your CAT will come from a comprehensive short-questions TEST held in the last practical period.
The CAT only accounts for 7.5% of your final course mark. Bearing this in mind, the test has been designed as a valuable revision exercise, rather than a source of easy marks. You should be ready to
"Provide brief notes or diagrams to answer the following 10 questions (all of equal value). Take care that your answers are relevant to the questions - marks will be deducted for answers which are wholly or partially irrelevant".
Time allowed 60-90 minutes. Some of the questions which were set in previous years are given below: seven of these questions will appear in the 1995 test.

a. Distinguish between: a) The diffusion constant and the diffusion coefficient. b) The pressure, partial pressure, and tension of a gas.

b. Draw diagrams showing the flow of air through the bird respiratory system during ventilation.

c. Use notes or diagrams to compare the effects of the level of oxygen and carbon dioxide on ventilation rate in freshwater and land animals. Briefly account for the difference.

d. Name the three respiratory pigments apart from haemoglobin. Indicate which of them is carried in cells, and suggest an advantage of this.

e. What are the major functions of: a) The aorta. b) Arterioles. c) Metarterioles. d) Capillaries. e) Capillary sphincters.

f. Draw a diagram showing the chemical mechanisms by which carbon dioxide is transported in the blood of mammals.

g. Draw a graph showing the changes of membrane potential and sodium and potassium permeability in an axon during an action potential.

h. Write brief notes on the major characteristics of: a) Inhibitory synapses. b) Electrical synapses.

i. Briefly describe the neural circuit for control of the contraction of a vertebrate skeletal muscle.

j. Draw diagrams to explain the structure and ventilation of the gills in a primitive mollusc.

k. Draw a diagram to show spiracular opening, and oxygen and carbon dioxide levels in tracheal gas, during cyclic respiration in an insect.

l. Note the effect of the following on the oxygen- dissociation curve of haemoglobin, and explain their significance: a) Temperature. b) Carbon dioxide.

m. Write brief notes on the major characteristics of: a) Giant axons. b) Myelinated axons.

n. Draw annotated diagrams to show how generator potentials of different sizes produce different frequencies of action potentials.

o. Give approximate figures for: a) The volume of oxygen in a litre of air. b) The volume of oxygen in a litre of water. c) The comparative solubility of oxygen and carbon dioxide in water. d) The comparative diffusion of molecules of oxygen and carbon dioxide in water. e) The comparative diffusion of oxygen and carbon dioxide into water from a partial pressure of 100mmHg.

p. Describe gas exchange in a sea urchin (echinoid).

q. Describe the structure of the photoreceptor cells of vertebrates (rods and cones).

r. Draw a diagram showing the major features of the heart and circulatory and respiratory systems of the African lungfish (Protopterus).

s. Describe the neural circuit for the knee jerk reflex in man.

t. Describe the nervous connections of the Mauthner cells of fish.

u. List (in order) the processes and membrane potentials involved in the conversion of stimulus energy to action potentials in a sensory neuron. Assume that there is a separate receptor cell, and accessory structures.

Fish obtain their oxygen from the water pumped across the gills. A fish in a sealed container of water will deplete the oxygen; if the concentration is measured before and after a known interval, the rate of oxygen consumption can be calculated. Water flows past the gills in one direction, due to the double pump system of mouth and opercular cavities. The rate of water flow depends on the need for oxygen (i.e. the activity level of the fish) and on the oxygen concentration in the water. A fish in a sealed container should thus increase its ventilation rate as it depletes its oxygen supply. In this practical, you will measure the oxygen concentration in aerated water before and after a fish has been sealed in it. You will also measure the ventilation rate of the fish, and of a control in an open dish, and so relate ventilation to oxygen availability.

Oxygen consumption of aquatic animals can be measured by several techniques, varying in complexity and the accuracy of results obtained. The Winkler method has been a standard for many years, and is often used in environmental studies. It is still one of the most accurate methods available, if done carefully; the only drawback is that it is relatively time- consuming. Manganous chloride and an alkali containing potassium iodide are added to the water sample. Manganous hydroxide is formed as a brown precipitate which absorbs the free oxygen in the water. After acidification with sulphuric acid, iodine is released in proportion to the oxygen content of the sample. The iodine is then titrated with sodium thiosulphate, using a starch indicator. As the free iodine is used up, the blue colour of the starch-iodine complex disappears.

Warning
Winkler reagents are dangerous: DO NOT PIPETTE BY MOUTH

Write-up
The difference in oxygen content between the water at the start and the end gives the amount of oxygen consumed by the experimental (flask) fish. Calculate the rate of oxygen consumption in ml g^-1 h^-1. Did the control fish also deplete the level of oxygen in the water ? Draw a graph of ventilation rate against time for both fish. Does the ventilation rate of the experimental fish increase, compared to the control, as its oxygen is used up ? Can you think of another mechanism which could explain this pattern ?

Reading
Fry, F.E.J. (1957). The aquatic respiration of fish. Chapter 1, pp 1-63, in The Physiology of Fishes, Part 1 ed. E. Brown. Academic Press, New York. QL 639 BRO

Water is a dense medium with a relatively low solubility for oxygen, characteristics which influence the respiratory organs of aquatic animals. Fish gills have evolved to present a large surface area for gas exchange, in a compact space, across which unidirectional water flow can be maintained. The surface area of the gills is related to the level of activity of the fish. The gill surface area is particularly low in air-breathing fish, which obtain part of their oxygen through accessory respiratory structures. The surface area of accessory structures is also likely to be low, in view of the high oxygen content of air. In this practical you will estimate the respiratory surface areas of the green-headed tilapia (Oreochromis macrochir), a moderately active fish, and Clarias gariepinus, a sluggish and air-breathing fish.

Work in pairs for this practical. Each student will dissect the respiratory organs of one side of either tilapia or Clarias, and then exchange fish with the other member of the pair.

Write-up
Present drawings of the gill structures of both species, and the completed results sheet. Explain how you calculated the rows marked * in your results section. Discuss how the areas of the different respiratory structures relate to the habits of the fish, and to the characteristics of gas exchange in water and air.

Blood and circulatory system of the frog

One of the most important functions of the blood is the transport of the respiratory gases oxygen and carbon dioxide to and from the tissues. The solubility of oxygen is low, but the carrying capacity of the blood is increased by molecules which combine reversibly with oxygen, taking it up at the respiratory surfaces and releasing it to the tissues. These molecules are proteins with metal ions, which give them complex patterns of optical absorbance. They are thus coloured, and so termed respiratory pigments.

The optical absorbance of the pigments differs between the oxygenated and deoxygenated states. This is of no functional significance to the animal, but is of use to the physiologist. If the absorbance spectrum of the pigment is known in both fully oxygenated and fully deoxygenated states, then the change in absorbance can be used as a measure of the percentage oxygenation of the blood. This information is used to construct oxygen dissociation curves, showing the oxygenation at different partial pressures.

In this practical you will examine the major features of the circulatory system of the frog and, if possible, obtain a sample of blood from the heart. The absorbance spectra of the haemoglobin of this blood, or sheep blood, will then be measured in both oxygenated and deoxygenated states.

Write-up
Present a clear, labelled drawing of your dissection, and brief notes on the functions of the vessels you have indicated. Present a graph of the absorbance spectra of oxyhaemoglobin and deoxyhaemoglobin from Xenopus. How do they compare with absorbance spectra of the respiratory pigments of other animals, and why ?

Calculation of resting potentials

This practical consists of a film showing some of the techniques used in electrophysiological experiments, and some calculations on the resting membrane potentials of excitable cells. Some aspects of the film will be discussed before it is shown. You may wish to take notes during this explanation, or during the film, to use in your write-up.

The calculations are designed to familiarise you with Nernst's and Goldman's equations. These should be done during the practical session, and signed in the usual manner. Give your results to the nearest 0.1mV.

1. The concentrations of ions in the cytoplasm of a mammalian skeletal muscle fibre and in the extracellular fluid were measured (mM):

Use the Nernst equation to calculate the equilibrium potential E (inside relative to outside) for each ion at 39^oC. (F = 96 500 Coulomb mol^-1, R = 8310 J K^-1 mol^-1)

2. The concentrations of ions in the cytoplasm of a squid giant axon and in squid blood were found to be (mM):

a) Calculate the equilibrium potential for each ion at 15^oC.
b) Use the Goldman equation to estimate the resting potential of the giant axon at 15^oC, given that the permeability (relative to potassium = 1.0) is 0.0013 for sodium and 0.0017 for chloride ions.

3. The concentrations of ions in a frog muscle fibre bathed in Ringer solution (physiological saline) were found to be (mM):

a) Estimate the resting potential of the muscle fibre at 0 ^oC, given that the permeability (relative to potassium = 1.0) is 0.01 for sodium and 0 for chloride.
b) Estimate the resting potential for different temperatures up to 35^oC, at 5^oC intervals.

4. The following results were obtained in an experiment where the resting potential of a frog muscle fibre was recorded (at 20^oC) in chloride-free saline with 80mM sodium and variable potassium concentrations:

For each potassium concentration, calculate:
a) The equilibrium potential for potassium, given that the cytoplasm has a potassium concentration of 140mM.
b) The resting potential of the muscle fibre, given that the sodium permeability is 0.01 that of potassium, and that the cytoplasm contains no sodium or chloride.

Write-up
Write a methods section for one of the experiments shown in the film; to test the effects of transmitter substances on a single cell in the brain of the cat.

Write results and discussion sections for the experiment described in exercise 4. The results should include a graph of membrane potential on log potassium concentration, showing
1. The measured resting potential (as in the table).
2. The calculated equilibrium potential for potassium.
3. The calculated resting potential.
The discussion should explain the significance of these results.

Receptors sensitive to chemical solutions directly applied are found in the insect orders Diptera, Lepidoptera, Hymenoptera and Coleoptera (Dethier, 1963). In the Diptera (flies) they are trichoid sensillae, hairs having three or four receptor cells, one of which is a mechanoreceptor. The other cells respond to sugars, salts, and water. The blowfly has chemoreceptors on the mouthparts and on the legs. Flies can thus sense surfaces on which they place their feet, and if suitable as food, will extend the mouthparts.

The extension of the mouthparts can be used as a behavioural indicator of the response of the tarsal chemoreceptors. Water only stimulates lowering of the mouthparts in a thirsty fly; a fly which has already drunk will not respond to water, or to sugar solutions too dilute to detect. A hungry fly will lower its mouthparts to detectable sugar solutions. The response to sugars can be prevented by salts in the solution, and so the sensitivity of both the sugar and the salt receptors can be measured. In this practical you will measure the sensitivity of the sugar receptors of the blowfly to different sugars.

Write-up
Present your results in tabular form. Discuss the sensitivity to different sugars in relation to the specificity of the sugar receptors and the diet of the blowfly.

References
Aidley, D. J. (1978). The physiology of excitable cells. 2nd edition. Cambridge: Cambridge University Press. QH 631 AID
Dethier, V. G. (1963). The physiology of insect senses. London: Methuen. QL 495 DET

The physiology of the nervous system cannot be understood without knowledge of its structure. In this practical, you will observe nerve tissue with the light microscope and measure and draw what you see. There are 7 slides for you to examine, draw, and meake measurements on. You must calibrate the graticule with the demonstration slide for each objective lens. The number of some slides is limited (e.g. x5), so use them whenever the opportunity arises. Give a scale bar on all drawings (rather than a magnification).

1. Spinal cord section (Turtox H10.311) (x7). The mammalian spinal cord has an inner region of neuron cell bodies (grey matter in unstained material), with an 'H' shape, surrounded by axons passing along the cord (white matter). The grey matter extends towards the dorsal and ventral roots, which are fibre tracts where sensory axons enter, and motor axons leave, the cord. There is a central canal in the grey matter, continuous with the ventricles in the brain, containing cerebrospinal fluid.

Draw the section under low power to show the distribution of grey and white matter. Draw areas of fibres and cell bodies under high power.

2. Motor neurons from spinal cord (Carolina H1660) (x22). This is a smear preparation, where the grey matter has been disrupted to separate individual neurons, and their dendrites; the axons have been broken.

Make high power drawings of two or three neurons. Measure the diameters of the cell bodies, and the lengths of the dendrites.

3. Nerve T.S. (UCR Rabbit sciatic nerve) (x2), or (UCR Rabbit, Weigert Pal) (x5), or (UCR Rabbit, Osmium) (x3)
Nerves consist of axons running in parallel. The axons are bundled together in fascicles, surrounded by a tough connective tissue perineurium. A nerve consists of one or more fascicles, surrounded by a diffuse connective tissue epineurium. In these sections the myelin sheaths are stained dark; unmyelinated axons are smaller and less obvious.

Draw the T.S. of the nerve under low power, showing the general features of its organization. Draw a group of axons under high power, showing both myelinated and unmyelinated fibres. Measure the diameters of 10 myelinated axons, and compare with diameters of a few unmyelinated axons.Compare these sections with T.S. Rabbit nerve Holmes (x2), which stains the cytoplasm, not the myelin sheaths.

4. Cerebellum silver stained (Carolina H1510) (x6). This part of the brain is involved in the coordination of movement. It has a complex structure, with an outer layer of cell bodies (the cortex), surrounding other regions of cell bodies and fibre tracts. A characteristic type of cell is the Purkinje cell, which has a large cell body at the base of the cortex, dendrites ramifying into the cortex, and a long descending axon.

Observe the organization of the cerebellum into cortex and inner fibre tracts and cell bodies. Measure and draw one or two Purkinje cells (you will not be able to identify much of the dendrite tree, or follow the axon, in this slide).

5. Neuroglia Fibrous astrocytes (Carolina H1420) (x4), or Protoplasmic astrocytes (Carolina H1423) (x4), or Microglia (Carolina H1426) (x4). Neuroglia are cells within the central nervous system other than neurons. They typically make up 90% of the cells, and 50% of the volume, of the brain, and have have functions in nutrition and mechanical support of the neurons. Different types of neuroglia are recognised, depending on their size and shape.

Measure and draw two or three glial cells of one of these types. A thicker dark-stained network may be present in the section: this is blood capillaries within the brain.

6. Pacinian corpuscle (Carolina H1688) (x2). A sense organ in the skin, sensitive to pressure. Other neurons in the skin are sensitive to touch, temperature, and pain, as seen in Part A; these are simply sensory nerve endings, often branched, but without accessory structures. Hair movements are detected by sensory nerve fibres wrapped around the hair follicles.

Draw the multilayered connective tissue capsule surrounding the sensory nerve fibre, and its position within the skin. Measure the size of the organ and the thickness of the connective tissue layers.

7. Motor end plate Motor nerve ending with plates (Carolina H1685) (x4), or Teased snake skeletal muscle stained with gold chloride (Carolina H1658, ). The synapse between a motor axon terminal and a skeletal muscle cell is known as a motor end plate. The axon loses its myelin sheath just before the end plate, which is a branched, beaded structure giving a large area of contact for transmitter release.

Make a high power drawing of a motor end plate and the associated axon and muscle cell. Measure the diameter of the muscle cell and the axon.

Write-up
Present a series of clearly-labelled drawings, noting the significant features of each section and the required measurements.