The Making and Un-Making of Neuronal Circuits in Drosophila

281 Pages·2012·5.046 MB·English

by  Liesbeth Zwarts, Jason Clements, Patrick Callaerts (auth.), Bassem A. Hassan (eds.)

N
EUROMETHODS
Series Editor
Wolfgang Walz
University of Saskatchewan
Saskatoon, SK, Canada
For further volumes:
http://www.springer.com/series/7657

The Making and Un-Making
of Neuronal Circuits in D rosophila
Edited by
Bassem A. Hassan
VIB Center for the Biology of Discase, Center for Human Genetics
VIB and University of Leuven School of Medicine
Leuven, Belgium
Editor
Bassem A. Hassan
VIB Center for the Biology of Discase
Center for Human Genetics VIB
University of Leuven School of Medicine
Leuven, Belgium
ISSN 0893-2336 ISSN 1940-6045 (electronic)
ISBN 978-1-61779-829-0 ISBN 978-1-61779-830-6 (eBook)
DOI 10.1007/978-1-61779-830-6
Springer New York Dordrecht Heidelberg London
Library of Congress Control Number: 2012939966
© Springer Science+Business Media, LLC 2012
All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the
publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA),
except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information
storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or
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The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified
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Cover Illustration Caption: Following traumatic incision, regenerating Drosophila circadian LNv neurons in the CNS
grow back to their original target sites and are seen approach the remnants of their former distal stumps (right top). In
this image the LNv neurons have been topographically colour coded and superimposed onto a Drosophila half brain
showing the optic lobe arborisation from the contralateral GFP expressing LNv neurons. This image was acquired with
a Zeiss Lumar V12 and a Zeiss LSM510 META NLO and processed with Adobe photoshop CS2. Image by Marta
Koch, Derya Ayaz and Bassem A. Hassan.
Printed on acid-free paper
Humana Press is part of Springer Science+Business Media (www.springer.com)
Preface to the Series
Under the guidance of its founders Alan Boulton and Glen Baker, the Neuromethods series
by Humana Press has been very successful since the fi rst volume that appeared in 1985.
In about 17 years, 37 volumes have been published. In 2006, Springer Science + Business
Media made a renewed commitment to this series. The new program will focus on methods
that are either unique to the nervous system and excitable cells or need special consideration
to be applied to the neurosciences. The program will strike a balance between recent and
exciting developments like those concerning new animal models of disease, imaging, in vivo
methods, and more established techniques. These include immunocytochemistry and
electrophysiological technologies. New trainees in neurosciences still need a sound footing
in these older methods in order to apply a critical approach to their results. The careful
application of methods is probably the most important step in the process of scientifi c
inquiry. In the past, new methodologies led the way in developing new disciplines in the
biological and medical sciences. For example, Physiology emerged out of Anatomy in the
nineteenth century by harnessing new methods based on the newly discovered phenomenon
of electricity. Nowadays, the relationships between disciplines and methods are more
complex. Methods are now widely shared between disciplines and research areas. New
developments in electronic publishing also make it possible for scientists to download
chapters or protocols selectively within a very short time of encountering them. This new
approach has been taken into account in the design of individual volumes and chapters in
this series.
Wolfgang Walz
v

Preface
The small fruit fl y, Drosophila melanogaster , has for over a century now had a large impact
on biological and biomedical research. From the description of the rules of heredity to the
elucidation of signals necessary for development, D rosophila research has greatly contributed
to—and often lead to—the emergence of important paradigms in our understanding of
development, physiology, behavior, and disease. The use of D rosophila as a model system
for the genetic control of almost every biological process one can think of has meant that
we now have a detailed understanding of the genetic logic underlying this animal’s
development, as well as of the cellular organization of most of its tissues and organs. It is
therefore rather surprising that our knowledge of the fl y brain has lagged signifi cantly
behind our understanding of other aspects of its development, physiology, and function. In
part, this is due to the sheer complexity of the brain. In that sense, despite a signifi cantly
reduced size in comparison to the mouse brain, for example, the fl y brain is exquisitely
complicated in its connectivity pattern and compartmentalization. Related to that is the fact
that studying the brain requires sophisticated tools, both genetic and technical, to carefully
dissect its development, physiology, function, and eventually degeneration and to eventually
link these different aspects to the genetic logic(s) underlying them.
The past decade has witnessed a massive expansion in the extent and sophistication of
the genetic tool kit of D rosophila . Because of the generic nature of genetic approaches,
brain research has benefi ted signi fi cantly from essentially all of these approaches. In addi-
tion, increasingly clever and technologically sophisticated behavioral assays have led to an
increase in the number of laboratories studying behavioral encoding using D rosophila as a
model system. Much more recent developments and expansions of these tools mean that
we are on the verge of a veritable revolution in our understanding of the genetic, cellular,
and network properties of the formation and function of the fl y brain.
This book is intended as a general introduction to the ideas and concepts behind the
methods, tools, and tricks that D rosophila neuroscientists utilize to decode the secrets of
their favorite enigma. It is by no means intended to be an exhaustive survey of the approaches
used in studying the fl y brain. Rather, it provides cutting-edge examples of the tools and
techniques used today in the research aimed at understanding the fl y brain as a model for
understanding how brains are organized and how they encode innate behaviors and learn
new ones. The major purpose is to equip current and future D rosophila neurobiologists with
a set of cutting-edge tools and the ideas underlying them so that fl y neurobiologists are able
not only to exploit these techniques but also to develop them further to suit their particular
needs.
In this sense, there are perhaps two aims of this volume that are essential to emphasize
in introducing it. The fi rst idea is that the major unifying focus of the different chapters is
the concept of a neuronal circuit, defi ned as a series of synaptically connected neurons sub-
servient to a particular behavioral modality. This is why, after an introduction to the general
anatomy of the fl y brain and its compartmental organization, all chapters dealing with ana-
tomical analysis focus on cellular and subcellular morphologies. These chapters range from
presenting the reader with currently available genetically encoded neuronal markers to
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viii Preface
methods for generating labeled single neurons, imaging them at very high resolution and
reconstructing those images, eventually into circuit diagrams. Although each of these chap-
ters uses examples in different neuronal lineages and at different developmental stages, the
basic tools and methodologies apply equally well to any neuron or circuit the reader wishes
to study. Once assembled, a circuit functions via synaptic communication between its mem-
bers in order to allow the animal to encode, produce, and learn behaviors. The classical
approach to circuit function is electrophysiological recording of neuronal activity. An
emerging, and increasingly powerful, alternative is visualization of synaptic activity through
genetically encoded indicators. P art II of the book provides two powerful examples of these
two approaches. Importantly, one chapter deals with studying neuronal physiology at larval
stages and the other in the adult brain. This is in order to cover the two major forms of the
Drosophila life cycle that are heavily used by researchers. In a very similar vein, Part III of
the book provides assays for larval as well as adult behavioral paradigms.
The second major aim of the book is to have a current and futuristic “feel” to it. To this
end, two elements were incorporated into the book’s concept. First, the choice of contribu-
tors is intended to re fl ect a relatively “young” group of fl y neurobiologists who are none-
theless at the forefront of developing tools for the study of brain development, anatomy,
and function. In fact, the lead authors of almost all chapters describe approaches that they
themselves have developed and that are setting new standards for neuronal circuit analysis.
Here, it is important to emphasize that there are many, many more such talented scientists
in the fi eld developing at least equally exciting approaches. However, it is simply impossible
to include the entire spectrum of tools currently available. Thus, this book by no means
claims comprehensiveness, but rather provides a relatively limited selection of many equally
good approaches, but which nonetheless covers a broad spectrum of modern concepts and
techniques. The choice of the contributors is also intended to provide methods that are
constantly being updated by the authors themselves. We hope that this allows the user
direct access to the source of further information about an insight into most of the meth-
ods. Second, we included a fi nal Part IV which includes chapters on relatively recent devel-
opments that are still fi nding their way into broader use and are still being further optimized
by many workers in the fi eld. Speci fi cally, we hazard to predict that more hardcore molecu-
lar, ex vivo and even in vitro approaches that have proven very powerful in gaining insights
into the working of mammalian neurons will become increasingly used in D rosophila neu-
robiology research to complement the classical in vivo genetic approaches. On the subject
of prediction, we also assume the risk of predicting that developmental, anatomical, physi-
ological, and behavioral vision research in D rosophila will likely rise to signi fi cant promi-
nence in the medium-term future of fl y neuroscience. This is why one of the two chapters
in Part II and both chapters of the behavioral section apply their tools strongly or exclu-
sively to the visual system. The fl y visual system has lagged behind other models mainly due
to the complexity and density of its connections. However, the rapid progress being made
at mapping the entire fl y brain connectome means that the complexity of the visual system
will change from being a foreboding into being an enticing problem to solve.
In closing, and on behalf of all the authors, we hope that readers will fi nd in this book
the information and tools necessary to carry out their current experiments and—more
importantly—further advance the progress of the D rosophila neurobiology fi eld and neuro-
biology in general.
Leuven, Belgium Bassem A. Hassan
Acknowledgments
We acknowledge Dr. P. Robin Hiesinger for Figure 10 and Dr. Carmen Francis for help in
establishing the in situ hybridization protocol. The work in the laboratory was supported
by grants from the IWT (Agency for Innovation by Science & Technology), FWO (grant
numbers G.0654.08 and G071412N) and VIB.
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