Isolated Central Nervous System Circuits

479 Pages·2012·13.344 MB·English

by  Stefan Trapp, Klaus Ballanyi (auth.), Klaus Ballanyi (eds.)

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

Isolated Central Nervous
System Circuits
Edited by
Klaus Ballanyi
Faculty of Medicine and Dentistry, Department of Physiology and Centre for Neuroscience,
University of Alberta, Edmonton, AB, Canada
Editor
Klaus Ballanyi
Faculty of Medicine and Dentistry
Department of Physiology
and Centre for Neuroscience
University of Alberta
Edmonton, AB, Canada
ISSN 0893-2336 ISSN 1940-6045 (electronic)
ISBN 978-1-62703-019-9 ISBN 978-1-62703-020-5 (eBook)
DOI 10.1007/978-1-62703-020-5
Springer New York Heidelberg Dordrecht London
Library of Congress Control Number: 2012944735
© Springer Science+Business Media New York 2012
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this
legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifi cally for
the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work.
Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the
Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions
for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution
under the respective Copyright Law.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not
imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and
regulations and therefore free for general use.
While the advice and information in this book are believed to be true and accurate at the date of publication, neither
the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be
made. The publisher makes no warranty, express or implied, with respect to the material contained herein.
Printed on acid-free paper
Humana Press is a brand of Springer
Springer 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 appeared in 1985. In about
27 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 which need special consider-
ation 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.
Neuherberg, Germany W olfgang Walz
v

Preface
Advances in methodologies and experimental models are pivotal to furthering our
understanding of central nervous system (CNS) functions in mammals. A most important
technology in that regard is “patch-clamp,” which was originally developed for monitoring
currents through single ion channels in “cell-attached” or “excised patch” confi gurations.
However, current neuroscience studies use patch-clamp primarily for the analysis of
membrane potential changes or underlying ion currents in the “whole-cell” mode, with
concomitant (defi ned) dialysis of the cytoplasm, while the “perforated” patch confi guration
can be applied to retain an intact cellular milieu. Patch-clamp techniques were adapted
about two decades ago for studying CNS cells in their natural in situ environment and have
since mostly replaced technically more challenging sharp microelectrode recording.
Similarly, exciting advances were established for optical approaches using fl uorescent dyes,
e.g., for monitoring membrane potential, mitochondrial potential, or cytosolic pH. Yet, the
most common optical approach is to visualize dynamic changes of the pivotal cytosolic
“second-messenger” Ca2 + , either in single CNS cells or neural networks that are comprised
of neurons and neighboring (micro)glia. Compared to initially used (charge-coupled
device) video cameras, optical spatial resolution is notably improved by confocal microscopy
while multiphoton imaging allows visualization of cells in deeper CNS layers. Besides,
infrared differential interference contrast optics are a convenient low-cost tool for visually
targeted patch recording in tissue depths of up to 150 μ m. Recently developed genetic
tools enable “knock-out” of particular cellular features, such as glutamate receptor subtypes,
or allow expression and subsequent imaging of intrinsically fl uorescent Ca2 + -sensitive or
structural proteins in identifi ed CNS cells. “Optogenetics” makes use of genetically inserted,
light-activated ion channels that change the activity of specifi c cells to reveal their functions.
Finally, local expression of cell-type-specifi c proteins is studied using immunohistochemical
approaches or molecular tools such as “western blots” or “polymerase chain reaction” for
analyzing cytoplasm of individual CNS cells, obtained by extraction after whole-cell
recording, or of homogenized nervous tissue.
Until about 30 years ago, most studies on mammalian CNS functions were carried out
using in vivo models of diverse mammalian species, mostly cats and rats, despite the fact
that various in vitro CNS models were developed already in the 1950s. It was not until the
mid-1970s that in vitro conditions were suffi ciently well developed to keep these isolated
CNS models, including cultures, en bloc (“slab”) preparations, and brain slices viable for
several hours in solutions that mimicked the composition of cerebrospinal fl uid (CSF) (or
rather the fl uid in the interstitial space within CNS tissues). At the same time, electrical
stimulation and single- or multiunit extracellular recording approaches plus sharp micro-
electrode intracellular recording techniques, such as “single electrode voltage-clamp,” were
adapted to these models. Until the end of the last millennium, work on acutely isolated
brain slices dominated CNS research with emphasis on studying mechanisms of synaptic
plasticity associated with long-term potentiation or depression evoked by afferent axon
tract stimulation. In these “classical” brain slices, electrical or pharmacological stimulation
vii
viii Preface
was typically needed for evoking neuronal responses, contrary to often pronounced spon-
taneous activity of the same cells in vivo. It was believed that this limitation is primarily
related to the fact that the thickness of brain slices is mostly less than 500 μ m for providing
suf fi cient diffusional supply of cells with the energy substrates oxygen and glucose con-
tained in superfused artifi cial CSF (ACSF). Consequently, neuronal dendrites and axons are
partly sectioned, which presumably attenuates network connectivity and thus depresses
spontaneous activity. To circumvent this, various laboratories used mainly newborn rodents
already since about 30 years ago to develop en bloc models with active neural networks.
During the same time period, others succeeded to keep large CNS aspects (up to entire
rodent brains) viable by arterial perfusion. During the last decade, procedures for generat-
ing active isolated CNS tissues have improved further, e.g., by using an ionic ACSF com-
position that refl ects more closely that of the fl uid in the extracellular space of neural
networks instead of that in CSF of the subarachnoid space a nd brain ventricles.
This contribution to the N euromethods series exemplifi es the application of a majority
of the above-mentioned and other technologies to mostly active in vitro preparations from
basically different CNS regions with a diversity of functions. Specifi cally, Chapter 1 by
Trapp and Ballanyi deals with neurons in rodent brainstem slices that control vagal outfl ow.
It outlines how “tonic” activity of these cells is modulated by metabolic processes and how
underlying mechanisms are studied with single channel and whole-cell patch-clamp tech-
niques, gas- and ion-sensitive microelectrodes, optical photomultiplier-based techniques,
and diverse molecular approaches. Further emphasis is on how methods for slice generation
and storage plus superfusate composition affect properties of these vagal neurons and neu-
rons in general. Chapter 2 by R uangkittisakul et al. delves deeper into the latter topic by
pointing out the particular importance of superfusate K+ , Ca2 + , and glucose, and also of
physical dimensions of newborn rodent en bloc and slice preparations for spontaneous
activity of respiratory neurons in the lower brainstem. Further, they exemplify that whole-
cell and suction electrode recording (for neuronal population activity) combined with mul-
tiphoton/confocal Ca2 + imaging is used for investigating contributions of neurons v ersus
glia to respiratory rhythm. Chapter 3 by Moore et al. summarizes techniques for studying
(spontaneous) activity in neurons of human fetal cortex slices, focusing on how slices of this
almost gel-like tissue can be generated and how developing electrical properties such as
immature Na + action potentials can be discriminated from imperfect whole-cell recording
conditions in these delicate cells. Chapter 4 by F ish et al. deals with histological character-
ization of physiologically determined fast spiking interneurons in slices of the dorsolateral
prefrontal cortex of monkeys. It outlines particularly how high-resolution confocal imaging
is combined with sophisticated optical analyses for elucidating structure-function relation-
ships for these cells. Chapter 5 by Nakamura et al. describes neural networks in the supra-
chiasmatic nucleus of the hypothalamus that continue to generate circadian rhythm in acute
slices or slice cultures. It shows how these circuits depend on experimental conditions, such
as time of day for their generation, and how they are analyzed with patch-clamp plus mul-
tiunit activity recording, molecular approaches, and Ca2 + or genetic bioluminescence imag-
ing. Chapter 6 by S tachniak et al. deals with other hypothalamic networks that regulate
plasma osmolarity in the body and whose responses to osmotic stimuli are studied with
patch-clamp and immunohistochemical approaches in conventional and thick hypothalamic
slices. In Chapter 7 by M cKay et al. , patch-clamp recording and histological analysis are
used to show that repetitive synaptic input establishes in vivo–like activity in rat cerebellar
slice neurons and that biophysical neuron properties change during postnatal development.
The latter fi ndings are important for comparing in vivo data from adult animals with in vitro
Preface ix
fi ndings that are often obtained in preparations from juvenile animals. Chapter 8 by S anchez-Vives
shows, using primarily whole-cell and multiunit activity recording, that patterns of sponta-
neous rhythmic activities in slices of adult cerebral cortex depend on animal species, super-
fusate composition, and temperature. Chapter 9 by B roicher and Speckmann reports how
spontaneous and evoked neuronal activities in acute cortical slices from patients who needed
surgical removal of brain tissue are analyzed by combining electrophysiological approaches
with voltage imaging. Chapter 10 by L uhmann and Kilb outlines how cellular properties
and network activity are analyzed in intact in vitro preparations of neonatal rodent cerebral
cortex. Chapter 11 by K antor et al. deals with the use of suction electrode recording and
Ca 2+ plus morphological multiphoton/confocal imaging for studying spontaneous network
oscillations in hippocampal, neocortical, and l ocus ceruleus slices from newborn rats and
piglets. Chapter 12 by De Curtis et al. describes methods for arterial perfusion of isolated
guinea pig brains that retain functional and interacting neural networks. Examples are
given for spontaneous and electrically evoked activities that are analyzed under normal
conditions and upon evoked seizures or ischemia with extra- plus intracellular electrophysi-
ological approaches, ion-sensitive microelectrodes, and voltage plus Ca2 + imaging. Chapter
13 by D ay and Wilson describes a juvenile rat model for independent dual perfusion of
carotid bodies and lower brainstem for analysis of contribution to respiratory rhythm of
peripheral and central chemoreceptors, respectively. Chapter 14 by B iggs et al. outlines how
to generate organotypic spinal cord slices for investigating with electrophysiologic and Ca2 +
imaging approaches mechanisms of pain-related central sensitization. Chapter 15 by
Mandadi et al. reviews slice and en bloc cord preparations for studying locomotor networks
with electrophysiologic and Ca2 + imaging approaches.
Due to space limitation, other established or recently developed isolated CNS prepara-
tions and their applications could not be dealt with, such as tonically active s ubstantia nigra
networks, isolated optic nerves or corpus callosum slices for studying axon-glia interactions,
or (organotypic) brain slices with intact connectivity of distinct regions, e.g., between the
thalamus and the cortex. However, the in vitro approaches and methodologies described
here are most likely applicable to further improve the latter models and to develop corre-
sponding models of not yet intensively studied structures such as n ucleus ruber , superior
colliculus , or basal ganglia.
Klaus Ballanyi