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THE AUSTRALIAN NATIONAL UNIVERSITY

RESEARCH SCHOOL OF BIOLOGICAL SCIENCES

DEPARTMENT OF NEUROBIOLOGY

ANNUAL REPORT 1971

STAFF

Professor:

G.A. Horridge, M.A., Ph.D., Sc.D.(Cantab.), F.R.S., F.A.A.

Fellow:

D.C. Sandeman, M.Sc.(Natal), Ph.D.(St.Andrews)

B. Walcott, B.A.(Harvard), PhD.(Univ. Oregon)

Research Fellows:

D. Young, B.A~(Oxon), Ph.D.(Wales)

L. Sosula, B.M.Sc., Ph.D.(Adelaide)

N.M. Tyrer, M.-A.-, Ph.D. (Cantab.)

I. A. Meinertzhagen, B.Sc. (Aberdeen), Ph. D. (St.Andrews)

E.E. Ball, A.B.(Stanford), Ph.D.(California)

Post Doe Fellow:

A. OkaJima D. Sci. (Tokyo)

Research Scholars:

R.G. Butler, B.Sc., M.Sc.(Tor.)

A. Ioannides, B.Sc. (Edinburgh)

J. Kien, B.Sc., M.Sc. (Syd.)

S.B. Laughlin, B.A.(Cantab.)

P.M.J. Shelton, B.Sc.(St.Andrews)

P.M. Thomas, B.Sc.(Flinders)

P .A. Dunn, B .A. (Oxon)

Honorary Research Fellow:

2.

J . S. Altrnan, :if .A. (Oxon), Ph . D. (London)

Research Assistants:

G. Stange, Ph. D. (Got tingen)

C. Giddings, B.Sc. (Exon)

Technical Officer:

R. Jackson.

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INTRODUCTION

There continues to be several main themes, all concerned

with the correlated structure and function of the nervous system.

The first is the analysis of various types of insect visual systems.

Although there is an apparently endless diversity of insect types,

we have reached the stage where the most important types are now

known in outline and a comprehensive review is in preparation.

Over the past year most of Professor Horridge's effort has been

upon mechanisms in eyes of nocturnal forms which make use of

special adaptations . to increase sensitivity.

It is also a continuing problem to find and bring into the

laboratory for analysis a sufficient number of large insects of

the particular groups still needed to complete the survey.

Walcott~ Butler, Ioannides and Meyer-Rochow are the ones

concerned with the - retina. Closely associated with this work

is the analysis of the neural processing of information in the

ganglia immediately behind the compound eye; Laughlin, Kien

are active in this area.

The second main theme is the analysis of the integrative

mechanisms which . control eye movement by the visual and statocyst

inputs in the crab, pursued by Sandeman, Okajima, Thomas and

Dunn. The third theme, perhaps the one now in the ascendancy,

is the analysis of mechanisms by which nerve fibres make the

correct connections with their target cells. This is the interest

of Ball, Tyrer, Young, Luff and Meinertzhagen.

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All the work involves an integration of results obtained

by a variety of techniques, basically interference microscopy,

electrophysiology of marked cells, electron microscopy, and

reconstruction from serial sections.

Clear-zone Compound Eyes

(G.A. Horridge with C. Giddings and G. Stange)

Insects that fly at night, and many crustaceans that live

in deep or muddy water, have a broad clear zone in the dark­

adapted eye between the optical system and the receptor part of

each ommatidium. As a result of work on several groups of insects,

three main functions for this clear zone are now recognized; as

a region where (a) a long light guide in each ommatidium can act

as an attenuator, (b) a composite image formed by many ommatidia

can be cast on the receptors, (c) scattered light can sum on the

receptors so that sensitivity is increased at the expense of

acuity. In the flour moth Ep~es tia evidently scattered light

is predominant, in the skipper butterflies an optical image

appears important while in nocturnal beetles the light guide

mechanism is supplemented by scattered light in the dark-adapted

state. As part of this work the anatomy of several clear-zone

eyes in Coleoptera and Lepi doptera has been described. It has

been necessary to devise a system for measuring the eyeshine of

the compound eye as seen from different directions to supplement

electrophysiological measurements of fields of view and

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behavioural tests of acuity.

Collaboration with Dept. Applied Maths.

(G.A. Horridge, Prof. B. Ninham and Dr. A. Snyder)

A fruitful collaboration has sprung up over the insect

eye as a system for mathematical treatment, so far in two aspects.

Prof. Ninham has solved the equations for the summation of

scattered light in the eyes of nocturnal insects and one result

is that a clear zone in the eye, with no sophisticated optics,

adds sensitivity by allowing scattered light to be summed on the

receptor layer. The other aspect is in the mathematical solution

of the modes of vibration in the light guides that occur in insect

eyes. There are many parallels with the theory of microwave

receiving aerials. Dr. A. Snyder is engaged on this work and

his theoretical treatment is matched by our experimental testing of

retinal function.

Crab Eye Movement

(D.C. Sandeman and A. Okajima)

When subjected to rotational stimuli, the stalked eyes of

crabs undergo nystagmus movements exactly similar to those of all

other animals with moveable eyes. It has been found that the eye

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movement system of the mud crab, ScyZZa serrata~ in semi-isolated

eye- brain preparations can be driven by artificaally irrigating

the statocyst (the equilibrium organ) with a fine jet of saline.

This provides a preparation in which the central neurones

involved in the eye movement system can be studied electrophysio­

logically. The study includes a characterization of the responses

of the equilibrium organ. This stage of the work is now

complete. One particular group of previously described receptors

are particularly sensitive to rotational stimuli and the

intensities of artificial stimulation necessary to evoke a

physiological response are now known. The investigation has also

shown how the fluid in the statocyst is affected by rotational

stimuli and confirmed that the artificial stimulus closely

resembles the natural stimulus.

Regeneration of Appendages in Crayfish.

(D.C. Sandeman and S.E. Luff.)

Arthropods have the ability to regenerate lost or amputated

limbs and re-innervate these correctly so that the new limb is

co-ordinated with other limbs. They therefore provide an

opportunity to study the remarkable specificity shown by

developing and regenerating neurons of all animals.

The decapod crustaceans are unique in that removal of the

stalked eye results in the regeneration of an apparently

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inappropriate organ. Instead of a new eye with optic ganglia,

an appendage resembling an antennule appears. Previous work

on this phenomenon has been sporadic and no attempt has been

made to produce enough animals with "heteromorph" antennules

for a careful study of the central connections of the new appendage.

A local species of freshwater crayfish Cherax destructor~

has been found to live well in the laboratory and will produce

a heteromorph . antennule up to Smm long 6 months after removal

of the eye. Two behavioural tests are used to determine whether

the . sensory input . from the heteromorph antennule is appropriate

t o an antennule or to an eye; (a) an attack response which is

specifically produced by stimulation of a normal antennule and

never by stimulation of the eye (b) a cleaning response directed

only at the eye and in which a particular pair of limbs are used.

Stimulation of heteromorphs of up to Smm in length (6 months

growth) will evoke only an avoidance response and occasionally

cleaning, but no clear attack.

It would appear that .the initial .sensory connections made

by the heteromorph . antennule . are more like those from tactile

receptors on the eye.

Inhibitory Slow Potentials in Crab Motoneurons

(D.C. Sandeman and P.A. Dunn.)

The unilateral withdrawal of the crab eye is produced by

just two large motoneurons. These can be found in living isolated

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brain preparations and penetrated with microelectrodes. Previous

work has shown that after the initial excitation of these neurons,

a strong repolarization of the post-synaptic membrane occurs. It

is thought that the repolarization is due to an inhibitory mechanism

and this and the excitation of the motoneuron are being studied by

applying -drugs to the central synaptic region of the motoneuron.

Structure and Physiology of the Yabbie (Crayfish)

Retina on Light Adaptation.

(B. Walcott)

The pigment .movements that occur in .the crayfish compound

eye on dark/light adaptation have been extensively described.

Also the structure of the retina has been described, but only at

the level of the light microscope. Therefore, this preparation

is analysed by combined intracellular recording and electron

microscopy. The . aim is to establish the anatomy of the retina

by electron microscopy and .the changes that occur in dark/light

adaptation, and .. the fields of individual photoreceptors that

accompany the structural changes.

So far the structural studies confirm early reports of

extensive pigment movements towards the middle of the retina

(the "clear zone") on light adaptation. The physiological studies

show that units of dark-adapted eyes are more sensitive to light

by 2-3 10g. units than light-adapted ones. Also dark-adapted units

have large angles of acceptance (about 15°) while light-adapted

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units have narrower angles (about 5°).

Mechanisms of Cell Movement and Intracellular

Pigment Movement in Arthropod Eyes.

(B. Walcott)

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In the compound eye of the giant water bug, Lethocerus, large

cell movements occur in dark/light adaptation. Preliminary

experiments indicate that depolarization of the receptors in the

absense of light may be sufficient to cause cell movements towards

the light-adapted state. This is being investigated.

Structural studies on the bug retina have shown highly

oriented microtubules which could be involved in the cell movements.

Studies are being initiated using drugs to disrupt microtubules

to clarify their role in these cell movements.

Similar studies are being done on the yabbie (Cherax destructor)

retina to determine the cause and mechanism of the extensive

intracellular pigment granule migration that occurs in dark/light

adaptation.

Motoneurons of the cockroach.

(D. Young)

In the present work, individually recognised nerve cells are

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being studied in the cockroach, Periplaneta americana~ with special

reference to the connections between nerve and muscle. Both the

motor neurons and muscles of this animal have been individually

ree0gnised and numbered in each segment. The connections of

individual motor neurons to their muscles has been determined by

intracellular study using microelectrodes filled with the dye

procion yellow. Intracellular stimulation demonstrates which

muscle the cell is connected to and dye is then injected from the

electrode to mark the cell body for subsequent identification

in histological sections. This technique is being used to study

the specificity of regenerating neuromuscular connections at

the level of single cells following various experimental alterations

to the system such as nerve transection and limb transplantation.

Development of the Sarcoplasmic Reticulum in the

Intersegmental muscle of the Locust Embryo

(N.M. Tyrer)

In contrast to developing vertebrate muscles which contain

a spectrum of cells at different stages of differentiation, cell

development in insect muscles tends to be synchronous, so that

at any stage of development the cells are at the same stage of

differentiation. This makes it feasible to relate the properties

of the developing tissue as a whole to changes occurring within

the cells.

Muscle contraction in the abdominal dorsal longitudinal

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muscles of locust embryos was related to ultrastructural changes

within the muscle. The time course of the mechanical response

changes as the muscles develop. Early in development both contraction

and relaxation rates are very slow and the muscle shortens very

little. Maximum contraction is achieved relatively early while

the rates of contraction and relaxation remain slow . Contraction

and relaxation become more rapid as the embryo develops but

rapid contraction develops earlier than rapid relaxation.

These changes can be related to the development of the

reticular system of the muscle fibres. When the muscle is first

able to develop tension, the myofibres contain scattered clusters

of myofilaments while the transverse tubule system (T system)

and sarcoplasmic reticulum (SR) are only poorly developed. At

the stage when full tension is developed the myofibres are full

sized and are packed with filaments but the T system is irregular

and the SR is still sparse . By the time the full contraction

rate is attained~ the T system and the SR are well developed

although there are fewer cisternae and the fenestrated reticulum

is not so profuse. About the time that the full relaxation

rate is achieved the cisternae and fenestrated reticulum become

more prominent.

Development of the Neural Control of Behaviour.

(N.M. Tyrer and J.S. Altman)

To investigate the specificity of contacts between nerve cells

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the normal sequence of events during the establishment of synaptic

connections must be understood. Developmental changes in embryos

are so comprehensive that it is difficult to isolate the neural

process associated with any particular behaviour pattern. Behaviour

appearing late in the life cycle is more suitable as development

is restricted to a few neurons. Insect flight is a suitable example.

The movement of the wings in the adult requires patterned activity

in the flight motor neurons which has not been necessary in the

nymphal stages. Using the Australian plague locust Chortoicetes

terminifera, we are approaching the problem in three ways.

(1) Development of flight behaviour (J.S. Altman)

In order to describe changes in individual neurons it is

necessary to identify the exact time at which the patterned

activity of the flight motor neurons first appears. Myograms

from the flight muscles show there are three phases: in the later

nymphal stages there is synchronous rhythmic firing in all flight

muscles. At the last moult the alternate firing of antagonistic

muscles suddenly appears and during the first five days of the

adult instar there is a steady increase in the frequency of the

firing pattern. The effect of the sensory input from the stretch

receptors in the last two phases is being investigated.

(2) Physiology of flight motor neurons (N.M. Tyrer)

Motoneurons to particular flight muscles have been identified

by intracellular recording, stimulating and dye injection.

Recordings from the four motoneurons to the main indirect wing

depressor muscle show electrical coupling between them and inputs

to them which lead to spiking at a frequency characteristic of

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of the flight patter n. Changes in the synaptic inputs to these

and antagonistic cells are being investigated over the critical

periods defined above.

(3) Morphology (J.S. Altman, N.M. Tyrer and A. Poyser)

The gross anatomical r elationships of cells and tracts in

the mesothoracic ganglion are being reconstructed. The morphology

of individual identified flight motoneurons is being described

using intracellular dye injection techniques and changes in their

processes during the maturation of the flight pattern is being

investigated. Synaptic inputs to known neurons are being

investigated using - electron dense .markers and electron microscopy.

Fibre Growth Patterns in the Developing Optic

Lobes of Calliphora .

(I.A. Meinertzhagen)

This is a study of the growth and development of certain of

the neuronal connections of the optic lobe of Calliphora. The

complex retina-lamina connections of ~he short retinula axons are of

particular interest because each axon terminates in one predictable

location out of very many other apparently identical ones. In

the third instar larva these axons grow between the eye imaginal

disc through the optic stalk and into the larval hemisphere,

where they terminate in the presumptive lamina. There is a

sequence of cell divisions in both the eye disc, forming retinula

cells, and the hemisphere, forming lamina monopolar neurones, and

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another sequence of axon growth between the two regions. By

studying the synchronisation of these patterns, of cell proliferation

and axon growth, it is possible to provide a rather exact description

of the minimal information which axons of each class need in order

to form their correct connections i.e. the actual contribution of

neuronal specification to the establishment of connections. Work

is in progress on the sequence of axon growth as reconstructed

from electron microscopy of portions of the growing optic stalk.

Further work will include the use of tritiated thymidine pulse

labelling to study the pattern of cell division in both the eye

disc and the developing lamina in order to define the cell lineages

of the different cell types.

Neuronal Structure and Function in the Thoracic

Ganglia of the Praying Mantis.

(E. Ball)

David Young of this laboratory has recently demonstrated that

serially homologous motoneurons in the meso- and metathoracic

ganglia of the cockroach control homologous muscles in the respective

legs and that the motoneurons of one thoracic segment will innervate

appropriate muscles in a leg transplanted from another segment.

Experiments now in progress are designed to reveal more information

about how regenerating nerves recognize, and re-establish

connections with, appropriate muscles.

I have started a comparable study on praying mantids

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(Archimantis sp.) in the hope that transplantation experiments

involving the highly modified prothoracic legs may be useful in

elucidating some of the rules by which nerves become appropriately

connected during development and regeneration.

However, before transplantation results can be properly inter­

preted, it is necessary to analyse the pattern of identifiable

nerve cells in the thoracic ganglia of the mantid and the innervation

of the highly modified prothoracic legs. Once this information has

been obtained it will be possible to proceed to transplantation

experiments, using them as tools to study neuronal specificity

and the origins of simple behaviour such as grooming.

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Publications.

BUTLER, R. 1971. The identification and mapping of spectral

cell types in the retina of Periplaneta americana. Z. vergl.

Physiol. ~' 67-80.

BUTLER, R. 1971. Very rapid selective silver (Golgi) impregnations

and embedding of invertebrate nervous tissue. Brain Research3

l1_, 540-544.

* GRADWELL, N. and WALCOTT, B. 1971. Dual functional and structural

properties of the interhyoideus muscle of the bullfrog tadpole

(Rana catesbeiana). J. exp. Zool. 176, 193-218.

HORRIDGE, G.A. 1971. Alternatives to superposition images in

clear zone compound eyes. Proc. Ray. Soc. Land. B. 179, 97-124.

HORRIDGE, G.A. and GIDDINGS, C. 1971. Movement on dark-light

adaptation in beetle eyes of the neuropteran type. Proc. Ray.

Soc. Land. B. 179, 73-85.

HORRIDGE, G.A. and GIDDINGS, C. 1971. The ommatidium of the

termite Mastotermes daruiniensis.. Tissue and Cell 3 l_, 463-476.

HORRIDGE, G.A. and GIDDINGS, C. 1971. The retina of Ephestia

(lepidoptera). Proc. Ray. Soc. Land. B. 179, 87-95.

IOANNIDES, A.C. and WALCOTT, B. 1971. Graded illumination

potentials from retinula cell axons in the bug, Lethocerus.

A. vergl. Physiol. ]l, 315-325.

LAUGHLIN, S.B. and HORRIDGE, G.A. 1971. Angular sensitivity of

the retina cells of dark-adapted worker bee. Z. vergl Physiol.

2!!._, 329-339.

* Not of this University.

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}lliYER-ROCHOW, V.B. 1971. Cataetyx memorabilis n.sp. ,-ein neuer

Tiefsee-Ophidiide aus dem sudostlichen Atlantik. Abh. Verh.

naturwiss. Ver. Hamburg~ (NF) li, 37-53.

MEYER-ROCHOW, V.B. 1971. Die seltenste fleischfressende Pflanze

Deutschlands: AZdrovanda vesicuZosa. Mikrokosmos~ ~, 29-31.

MEYER-ROCHOW, V.B. 1971. A crustacean-like organization of insect

rhabdoms. CytobioZogie~ i, 241-250.

MEYER-ROCHOW, V.B. 1971. Fixierung van Insektenorganen mit Hilfe

eines Netzmittels - Das Dorsalauge der Eintagsfliege AtaZophZebia

costaZis. Mikrokosmos~ 60, 348-352.

MEYER-ROCHOW, V.B. 1971. Endemische Tiefsee-Organismen vor

Slidafrikas Westkliste. Namib und Meer~ ~, 59-63.

MEYER-ROCHOW, V.B. 1971. 'Nashi' -die Apfelbirne (Pyrus serotina

Rehder) aus Japan. Mitt. Obstbauversuchsring~ ~, (12), 446-452.

MEYER-ROCHOW, V.B. 1971. Observations on stridulating Australian

beetles (HydrophiZidae~ Cerambycidae~ PassaZidae~ Dynastidae).

Forma et Functio, 1, 326-339.

SANDEMAN, D.C. 1971. The excitation and electrical coupling of

four identified motoneurons in the brain of the Australian

mud crab, ScyZZa serrata. Z. vergZ. PhysioZ. ~. 111-130.

SANDEMAN, D.C. and MENDUM, C.M. 1971. The fine structure of

the central synaptic contacts on an identified crustacean

motoneuron. Z. ZeZZforsch. 119, 515-525.

SHELTON, P.ll.J., HORRIDGE, G.A. and MEINERTZHAGEN, I.A. 1971.

Reconstruction of synaptic geometry and neural connections from

serial thick sections examined by the medium high voltage electron

microscope. Brain Res. ~, 373-377.

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WALCOTT, B. 1971. Cell movement on light adaptation in the

retina of Lethocerus (Belostomatidae, Hemiptera). Z. vergZ.

PhysioZ. li, 1-16.

WALCOTT, B. 1971 . Unit studies on receptor movement in the retina

of Lethocerus (Belostomatidae, Hemiptera). Z. vergZ. PhysioZ.

li, 17-25.

WALCOTT, B. and HORRIDGE, G.A. 1971. The compound eye of

ArchichauZiodes (Megaloptera). Proc. Roy. Soc. B.~ 179,65-72.