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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.