Paxinos and Franklin's the Mouse Brain in Stereotaxic Coordinates, Compact: The Coronal Plates and Diagrams

By Keith B.J. Franklin and George Paxinos

Paxinos and Franklin's The Mouse Brain in Stereotaxic Coordinates, Compact Fifth Edition, is the compact version of the most widely used and cited atlas of the mouse brain in print. It emulates in design and accuracy Paxinos and Watson’s The Rat Brain in Stereotaxic Coordinates, the most cited publication in neuroscience. The compact edition provides the coronal plates and diagrams of the full mouse atlas in a smaller, more convenient spiral format and at a student friendly price. High resolution digital photographs of the coronal plane of section from the full 5th edition complement the coronal drawings. Unique to the compact, it includes an introduction to the use of the atlas in stereotaxic surgery.

• Contains 100 coronal diagrams that were fully revised for this new edition
• Includes 100 coronal photographic plates produced from directly scanned, very high-resolution images of the biological sections (done at the Allen Institute)
• Provides a beginner's guide with 25 pages on conducting stereotaxic surgery and how to use the atlas
• Presents surface views of the brain with labels over the major structures
• Uses the best ontology tree (nomenclature based on the development of the brain) with universal applications across mammals

• Language: English
• Publisher: Academic Press
• Release date: May 19, 2019
• ISBN: 9780128161609

Keith B.J. Franklin

Dr. Franklin is Professor Emeritus at McGill University in the Department of Psychology. He is interested in neural mechanisms of motivation, particularly the role of specific neurotransmitter systems. His research uses pharmacological and molecular biological methods to study the role of monoamines, opiate peptides and neurosteroids in pain, memory and drug dependence.

Book preview

Paxinos and Franklin's the Mouse Brain in Stereotaxic Coordinates

Compact 5th Edition

Fifth Edition

George Paxinos

Neuroscience Research Australia and The University of New South Wales Sydney, Australia [e-mail]

Keith B.J. Franklin

Department of Psychology McGill University Montreal, Quebec Canada H3A 1B1

Table of Contents

Cover image

Title Page

Copyright

Dedication

Preface

Reproduction of Atlas Figures in Other Publications

Acknowledgements

Key features of the Compact Fifth Edition

Introduction

Surgery

Histology

Preparation of Images and Drawings

Coronal, Sagittal, Horizontal Planes and an Example

Ontology, Nomenclature and Abbreviations

The Basis of Delineation of Structures

Telencephalon - Pallium

Parts of the Mouse Brain

Parts of the Mouse Brain – 2

Parts of the Mouse Brain – 3

Principles of Stereotaxic Positioning

Preparation for Stereotaxic Surgery

Inserting the Mouse in the Stereotaxic Apparatus

Exposing the Skull Surface and Confirming a Skull Flat Position of the Head

Implanting an Electrode or Canula

List of Structures

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

R

S

T

U

V

X

Z

Index of abbreviation

Figures

Figures

Copyright

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Notices

Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary.

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility.

To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein.

Library of Congress Cataloging-in-Publication Data

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British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library

ISBN 978-0-12-816159-3

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Publisher: Natalie Farra

Acquisition Editor: Natalie Farra

Editorial Project Manager: Kathy Padilla

Production Project Manager: Andrew Riley

Cover Designer: Matthew Limbert

Typeset by SPi Global, India

Dedication

Dedication: To Grace and Fran

Preface

Atlases, like theories, assist you to find your way in unknown domains.

In the 22 years since the first edition of The Mouse Brain in Stereotaxic Coordinates, there has been increased interest in the mouse in neuroscience. No mouse is an island. The mouse is studied for its relevance to human health. In the present atlas, we are using the same nomenclature and abbreviations as for our human brain atlas (Mai et al, 2015). This way, inspired by human conditions, scientists can navigate seamlessly between human and mouse to test hypotheses and then relate their observations to the human.

Reproduction of Atlas Figures in Other Publications

As authors, we give permission for the reproduction of any figure from the atlas in other publications, provided the atlas is cited. Permission from the publisher should be sought on-line via the Elsevier homepage (http://elsevier.com/locate/permissions) or from Elsevier Global Rights Department in Oxford, UK: phone: (+ 44) 1865843830, fax: (+ 44) 1865 853333, email: [e-mail].

How to cite this atlas: Paxinos G and Franklin KBJ, Paxinos and Franklin’s The Mouse Brain in Stereotaxic Coordinates, Compact 5th Edition, San Diego, Elsevier Academic Press, 2019.

Acknowledgements

We have had the support of a talented editor, Dr Natalie Farra of Academic Press. We thank Yvette Paxinos (http://www.paxifilm.com) for the cover design, Charles Watson for the skull diagram, Emma Schofield for the use of the image for the cover, Fran Abbott for the design of diagrams and Dan Binks for the index. This work was supported by NHMRC grants (APP1086083, APP1086643), the ARC Centre of Excellence for Integrative Brain Function (CE140100007) and an NIH BRAIN Initiative award (U01MH105971).

Key features of the Compact Fifth Edition

•100 thoroughly revised coronal diagrams and accompanying photographic plates spaced at approximately 120 μm intervals

•Photographic plates printed from high resolution digital images in color

•The most accurate and virtually universally used stereotaxic coordinate system

•Over 800 structures identified

•Electronic diagrams available to purchasers of this book via a password-protected web site

Introduction

The brain depicted was the most complete in a sample of brains from 26 adult (3 month old) C57BL/J6 mice (weight range 26-30 g). The choice of mouse strain and weight range presented some difficulties because mouse strains vary much more in weight and brain anatomy than rat strains (see Wahlsten et al., 1975). The C57BL/J6 strain was chosen because it is one of the most widely used and is of intermediate size. Stereotaxic surgery on the mouse is relatively difficult because of fragility of the mouse skull. We, therefore, chose a weight range representative of the fully adult mouse in which the skull is calcified and presumably stronger.

Some adjustments for use of this atlas with other strains can be calculated from Wahlsten et al. (1975) who show stereotaxic grids in the sagittal plane for seven strains of 77- to 78-day-old mice, with the same skull flat orientation as this atlas.

Plates are usually 120 μ apart, except where no usable sections were available; the inter-plate distance is as small as 40 μm or as large as 160 μm. Alternate sections were stained with cresyl violet or were reacted to reveal acetylcholinesterase (AChE). Intermediate sections were processed immunohistochemically to reveal parvalbumin. These sections were used to aid delineations, but are not reproduced in the atlas. Other histochemical markers used to assist delineation (using different brains, but not shown in the atlas), include immunohistochemical stains for substance P and tyrosine hydroxylase, Timm’s reaction for zinc, the NADPH-diaphorase stain for nitric oxide synthase and the iron-hematoxylin stain for myelin.

Surgery

All procedures involving live animals were carried out in accordance with accepted ethical principles for animal research and were approved by the relevant Animal Ethics committees of The University of New South Wales and McGill University.

While under sodium pentobarbital anesthesia, the mice were placed in a Kopf small animal stereotaxic instrument. The head was positioned by means of a mouse nose clamp adaptor (Kopf Model 922) supplemented by rat ear bars (Kopf Model 957) placed lightly in the external auditory meatus to locate the interaural line. Because the skull of the mouse is extremely fragile and easily compressed, the Kopf mouse holder is designed to be used without ear bars (Slotnick and Brown, 1980). If ear bars are used they must be inserted with only a few grams of pressure or else the bones of the external auditory meatus or the skull will be crushed. Ear bars can be used successfully if they are inserted so as to merely touch the meatus, but any further pressure may lead to breathing difficulties.

The position of the head was adjusted so that the height of the skull surface at bregma and lambda was the same. Lambda was defined as the point of intersection of the sagittal suture and the line of best fit along the lambdoid suture (see skull diagram). The mean (± SD) position of bregma was 3.8 (± 0.25) mm rostral and 5.8 (± 0.48) mm dorsal to the interaural line (IA). Lambda was located 0.41 (± 0.26) mm caudal and 5.8 mm dorsal to the interaural line. These values were used to define the scaling of distances on the stereotaxic grid.

To establish the stereotaxic position of brain structures, reference needle tracks were made perpendicular to the horizontal and coronal planes and at predetermined distances from bregma and the interaural line (AI; vertical tracks: 2 mm anterior to bregma and 1 mm anterior to the IA; horizontal track: 3 mm above the IA; all tracks were 1.5 mm lateral to the midline).

The coronal set of the first edition reached only the level of the area postrema. For the second edition we inserted another 7 levels obtained from a different mouse of the same strain and weight.

Histology

After surgery, while still under anesthesia, mice were killed by transcardial perfusion of 20 ml ice-cold phosphate buffered saline (0.1 M PBS, pH 7.3), followed by 20 ml of 4% paraformaldehyde in PBS. The brain was immediately dissected free from the skull and placed dorsal surface down in a small aluminum foil boat containing 1.5% gelatin dissolved in 0.9% saline solution. The aluminum foil boats were made by folding aluminum foil around an appropriately sized rectangular block (20 x 12 x12 mm). The brain was oriented in the boat so that its anterior-posterior axis was parallel to the long axis of the boat. When suspended in the gelatin solution, the brain floats so that its dorsal surface is almost exactly parallel to the flat skull horizontal plane. When the brain was correctly positioned, the boat was placed in a refrigerator for 20 min for the gelatin to jell. The sides of the aluminum boat were then folded down to expose the gelatin block. The foil now formed a tray that was used to lower the gelatin block into dry ice-cooled isopentane. The block was lowered slowly so that freezing proceeded rostrally through the brain, from brainstem to olfactory bulb and no more than 1-2 mm of unfrozen tissue was below the surface of the isopentane at any time. The frozen block was then stripped of the remaining aluminum foil and attached to the chuck of the microtome with mounting medium. The planar sides of the rectangular block were used to orient the block on the chuck, with the anterior-posterior axis of the brain perpendicular to the surface of the chuck. Only minor adjustment of the orientation of the chuck in the microtome was then necessary to cut sections perpendicular to the horizontal and vertical stereotaxic planes.

The brain was cut at 40-μm thick sections. Unattached gelatin was brushed away and the section was taken up into a drop of PBS on the surface of an acid-cleaned and gelatinized slide. Out of every three successive sections (4 for the horizontal plane), two (3 for the horizontal plane) were taken onto a slide designated for staining of Nissl or AChE. If the first two sections were deemed satisfactory, the third was set aside in ice-cold PBS for parvalbumin immunohistochemistry. The following three sections were designated for staining for AChE or Nissl, as per the protocol of Paxinos and Watson (1986, 2007). A detailed protocol of the staining procedures can be found at http://www.neura.edu.au/research/themes/paxinos-group (1/3 of the way down on the right you will find Nissl and AChE Staining Protocols for Beginners). Our aim was to reliably obtain precisely oriented sections of relatively undistorted mouse brain in which Nissl- and AChE-stained sections alternated. Fresh or lightly fixed mouse brain tissue is so soft that it distorts under its own weight, but it assumes its normal shape when supported in a viscous fluid. Jelled gelatin solution adequately supports the brain during freezing, but the gelatin causes the sections to curl when they are cut in a freezing microtome. The addition of sodium chloride to the gelatin solution promotes the formation of fine crystals in the gelatin block so that the embedding material shatters when sectioned and can be gently brushed away from the frozen sections. Freezing must proceed steadily through the long axis of the block. If the whole block is placed directly into the cooling medium, the outside of the block freezes and forms a rigid shell so that when the brain expands during freezing, it distorts. Sections were taken up into a drop of PBS to eliminate minute bubbles which become trapped under the section if sections are thawed directly onto a slide. With partially fixed mouse brain, the minute bubbles perforate the section when it dries.

Preparation of Images and Drawings

Photographs of selected sections were made onto 4 x 5 inch Kodak Plus X or AGFA negatives using a Nikon Multifid photomicroscope and 20 x 22 inch black and white prints were made of these negatives on AGFA multigrade photographic paper. The delineations and fiducial marks were hand drawn onto acetate tracing paper placed over the photographs. The tracings were scanned and the resulting images were used as templates for tracing using Adobe Illustrator. For the reproduction of plates as well as for the web site, we used images obtained at the Allen Institute by their excellent Microscopy Unit. Approximately 100 images were taken per section and they were stitched together.

It was thought that the drawings would be more informative if they were not stylized and for this reason, with the exception of small adjustments to distorted midlines and cortex, the drawings depict the asymmetries present in the sections. When part of a section was missing or severely distorted, it was drawn in after consideration of sections obtained from other brains.

Skull Diagram

Dorsal and lateral views of a mouse skull. Bregma and lambda are the intersections of the midline suture with the line of best fit along the bregmoid and lambdoid suture, respectively. Diagram curtesy Charles Watson.

Coronal, Sagittal, Horizontal Planes and an Example

The large number at the bottom left of each figure shows the anteroposterior distance of the corresponding plate from the vertical plane passing through the interaural line. The large red number at the top right shows the anteroposterior distance of the plate from bregma. The numbers on the left margin show the dorsoventral distance from the horizontal plane passing through the interaural line. The red numbers on the right margin show the dorsoventral distance in mm from the horizontal plane passing through bregma and lambda on the surface of the skull. The numbers on the top and bottom margins show the distance in mm of structures from the midline.

As an example, if it is necessary to insert an electrode into the ventromedial hypothalamic nucleus, Fig. 44 reveals the coordinates with reference to the interaural line to be 2.22 mm anterior to the interaural line, 0.25 mm dorsal to it and 0.4 mm lateral to the midline. The coordinates of the same structure obtained with reference to bregma are 1.58 mm posterior to bregma, 5.5 mm ventral to it and 0.4 mm lateral to the midline. The location of bregma and lambda on the mouse skull is shown in the skull diagram.

No atlas or stereotaxic instrument will compensate for using bregma and lambda points inappropriately. These reference skull marks are the midpoints of the curve of best fit along the coronal and the lambdoid suture, respectively. They are not necessarily the points of intersection of these sutures with the midline suture.

Researchers usually wish to block the brain at the same coronal (or sagittal) plane as the atlas plane (flat skull position) so that they can determine most readily the location of their electrode placements and identify structures according to the atlas. For better results they need a brain blocker (matrix). After blocking, the brain needs to be placed on a chuck with surface parallel to the cryotome knife. For cryotomes without zero-tilt position (what a pity they do not have it), this can be achieved by freezing some mounting medium directly onto the chuck and cutting through the mounting medium to create an aligned surface on which to position the blocked tissue.

Ontology, Nomenclature and Abbreviations

Progress in brain research has been hampered because of the lack of consensus on ontology, nomenclature and a canonical set of abbreviations. As it concerns ontology, there is now reason to adopt a new, evidence-based ontogenetic ontology. Puelles et al (2013) published the theoretical rationale for such an ontology and this is further explained in the Puelles et al second edition of The Chick Brain in Stereotaxic Coordinates (2019). Fear not for it being a chick. Birds have first class brains, they just have not had good public relations.

There is an obvious need for a stable neuroanatomical nomenclature to accurately and efficiently convey information between neuroscientists. Despite this, many terms and abbreviations are still used in the literature to describe a single structure. Neuroscience communities concerned with different systems have developed identical abbreviations for completely different structures; for example, SO may stand for both supraoptic nucleus and superior olive, SC for suprachiasmatic nucleus and superior colliculus and IC for inferior colliculus and internal capsule. Further, homologous structures are nonetheless named or abbreviated differently in different species.