Atlas of the Neonatal Rat Brain 1st Edition

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 Dedication

We dedicate this scientific work to our parents who inspired us to follow our interests
and provided unwavering support to us during all our struggles and successes.

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 Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

About the Authors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xix

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiii

Section Iâ•… P-1 Brain

Coronal Plates (Figures 1 through 30) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Sagittal Plates (Figures 31 through 44) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Section IIâ•… P-7 Brain

Coronal Plates (Figures 45 through 71) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Sagittal Plates (Figures 72 through 95) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Section IIIâ•… P-14 Brain

Coronal Plates (Figures 96 through 136) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

Sagittal Plates (Figures 137 through 157) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

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 Preface

T he rat brain develops enormously after birth and the changes are very rapid. We
have attempted to prepare a photographic atlas representing the neonatal rat brain at
postnatal (P) days P-1, P-7, and P-14. This atlas illustrates the main anatomical features
at these three ages.
It will serve the needs of researchers and students who are interested in postnatal
development, slice cultures, developmental disorders, neuroanatomy, neuropharmacol-
ogy, neurobiochemistry, and neuropathology of rats. We hope that this atlas will provide
a template for comparative studies with other species and numerous animal models of
brain pathology in rats.

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 Acknowledgments

W e are grateful to the following individuals for making this atlas work possible
through their moral, emotional, technical, and scientific support: David Good,
Keith Elmslie, Kala Venkiteswaran, Sriram S. Shanmugavelandy, Christopher Lieu,
Anand Rao, Mathew Berk, Timothy Gilmour, and Barbara Norwitz. We also acknowl-
edge financial support for this work from research grants to Thyagarajan Subramanian
from the National Institutes of Health (NS42402), Commonwealth of Pennsylvania
Tobacco Settlement Biomedical Research Fund, and the Penn State University Brain
Repair Research Fund. We also thank the U.S. Department of Health for the Physician
Scientist Research Award.

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 About the Authors

Renuka Ramachandra, is a postdoctoral researcher at the AT Still University in

Kirksville, Missouri. She received her PhD degree in neurophysiology from India and
her initial postdoctoral training at the National Brain Research Centre in India, a premier
neuroscience institute. Dr. Ramachandra worked on the effect of barrel cortex lesions in
rats and on the effects of drugs on the developing rat brain. She also worked as a research
scientist in the embryonic stem cell group at Reliance Life Sciences, Mumbai, India,
where she differentiated human embryonic stem cells into oligodendrocytes for spinal
cord injury treatment. She went on to use her skills and knowledge of basic electrophysi-
ology, brain anatomy, and histological techniques to develop an in vitro basal ganglia
slice culture model in the Department of Neurology at Penn State College of Medicine.
This model is used to study in vitro cell transplantation. During the development of this
model, she recognized the need for a neonatal rat brain atlas. Dr. Ramachandra created
this atlas for her project and decided to publish it so that it would be available to all neu-
roscientists and students who need one.

Thyagarajan Subramanian is a professor of neurology and neural and behavioral

sciences at Penn State University Hershey Medical Center in Hershey, Pennsylvania.
Dr. Subramanian received his initial medical training from Calicut Medical College in
Calicut, India and his graduate training in developmental neurobiology and neural cell
transplantation under the guidance of Dr. R. D. Lund at the University of Pittsburgh
School of Medicine, Pittsburgh, Pennsylvania. He completed his neurology residency
at the University of Pittsburgh followed by fellowship training in neurological research
and movement disorders at Emory University in Atlanta, Georgia under the guidance
of Drs. Mahlon R. DeLong, Ray L. Watts, and Roy A. E. Bakay. Dr. Subramanian’s
research interests are in developmental neurobiology, basal ganglia physiology, cell
transplantation, gene therapy, and experimental neurotherapeutics. Using a variety of ani-
mal models, cell and tissue culture techniques, in vivo electrophysiology, and detailed
histology, Dr. Subramanian and co-workers have described several novel findings in
central nervous system (CNS) response to transplantation. Among his major scientific
contributions are his work on creating an MRI based brain atlas of the Rhesus monkey,
description of the effects of immunomodulation on host responses to CNS xenografting,
and the description of the use of retinal pigment epithelial cell grafts for CNS repair.
Dr. Subramanian has directed the medical neuroscience course for medical and gradu-
ate students and trained numerous medical students, as well as neurology, neurosurgery,
physiatry, psychiatry, and geriatric residents. He has served as the education committee
chair of the American Society for Neural Therapy and Repair (ASNTR) and has trained
numerous scientists and physicians who now work as independent investigators in many
institutions all over the world.

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 Introduction

R odents have frequently been used to model a number of neurological disorders. The
neonatal rat in particular has been used in many developmental studies and exten-
sively for preparing slice culture models that permit the study of interactions among dif-
ferent regions of the brain during development. Neonatal rats have also been used as
models for neurotoxicological screening and mechanistic studies (Becker and Liu, 2006;
Noraberg, 2004). During our attempts to develop a neonatal rat brain slice culture model
of basal ganglia development, we realized that there was no published neonatal rat brain
atlas. This took us by surprise as we knew of quite a few adult rat brain atlases. To rem-
edy this situation, we developed this atlas showing representative development of the rat
brain between postnatal day 1 (P-1) and postnatal day 14 (P-14). A number of animals
from each litter were euthanized on the designated postnatal dates and the brains were
sectioned, stained, photographed, and annotated to prepare this atlas.
This atlas is an effort to provide a guide to the neonatal rat brain. It contains a com-
prehensive set of histological images of the newborn rat brain from P-1, P-7, and P-14.
Although we prepared brain sections for all the ages from P-1 to P-14, we have chosen to
present these three ages as representative to provide a template of developmental matura-
tion of the neonatal rat brain at various stages. Further, the inclusion of every age between
P-1 and P-14 would needlessly add pages to this atlas without adding value. Additional
images will be made available as electronic resources for individuals who seek images
not represented in this volume. P-0 was covered earlier in The Atlas of the Developing
Rat Nervous System€(Paxinos et al., 1994), so we started at P-1, proceeded to P-7 (the
midpoint in neonatal development), and concluded at P-14. This atlas contains both coro-
nal and sagittal sections for all the three age groups. The P-1 section contains 30 coronal
plates and 14 sagittal plates; P-7 includes 27 coronal plates and 24 sagittal plates. The final
P-14 section shows 41 coronal plates and 21 sagittal plates. Each set consists of contiguous
sections from individual animals with no substitutions or omissions. The sections were
prepared carefully to ensure that their orientations were maintained and were consistently
of the highest quality. The selections were based on the structural variability represented.
Care was taken to minimize tears and distortions. Fixation and staining cause minimal
amounts of shrinkage and damage to tissues. However, we feel that the structural details
are well preserved.
This atlas has certain unique features. The sections are Nissl stained with cresyl vio-
let—the most common staining technique used in the neurosciences. Future editions will
include specific immunostains and special stains in the same format as these Nissl stains.
The photomicrographs achieve high resolution and clarity. The structures are directly
labeled on the images, making it easier for readers to correlate data. The electronic ver-
sion will allow labels to be removed so the atlas can be used as a teaching tool.

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 xvi Atlas of the Neonatal Rat Brain

Animal Preparation
Sprague-Dawley rat pups were used for this work. Pregnant dams from Charles River
Laboratories (Wilmington, MA) were received on day 13.5 of gestation and housed in
standard laboratory conditions, with 12-hour dark and light cycles and administration
of food and water ad libitum. All procedures complied with guidelines issued by the
National Institutes of Health and were approved by Penn State University’s Institutional
Animal Care and Welfare Committee. After birth, rat pups ranging in age from P-1 to
P-14 were sacrificed at the same time every day. The sexes of the newborns were not taken
into consideration. The neonates were decapitated and the brains were removed carefully
and postfixed in 4% paraformaldehyde for 3 to 4 days. This allowed preservation of the
brain structures and made it easier to isolate the brains from the delicate skulls. The
brains were then cryoprotected in 15% sucrose in phosphate buffered saline (PBS) and
then in 30% sucrose in PBS.
The brains were positioned and frozen on sucrose blocks made on the base of a slid-
ing microtome that was then used to cut the brains at 50-µm thickness for both coronal
and sagittal views. For sagittal sections, each brain was cut along the midline carefully
and placed with the midline facing the block. Care was taken to retain and mount every
section cut. We noted all missing sections so that we could calculate the correct distance
from the midline for the sagittal sections.

Section Processing
Every section was mounted on polylysine-coated slides, air dried, and stained for Nissl
bodies using cresyl violet. In short, the slides with the sections were passed through the
solutions for 3 minutes each in the following order except where noted: 100% ethanol,
100% ethanol, 95% ethanol, 75% ethanol, water (single dip), cresyl violet (3 to 4 min-
utes), water, 75% ethanol, 95% ethanol, 100% ethanol, 100% ethanol, and xylene. The
slides were cover slipped using DPX mounting media and left to dry for 2 days before
image capture.
To make 500 ml of 0.5% cresyl violet (pH about 3.9), we mixed 2.5 g cresyl echt violet,
300 mL water, 30 mL 0.1 M sodium acetate (13.6 g granular sodium acetate in 92 mL
water), and 170 mL 1.0 M acetic acid (29 mL glacial acetic acid added to 471 mL water).
This solution was mixed at least 7 days on a magnetic stirrer and then filtered.

Imaging
Using Neurolucida software (Version 8, MBF Biosciences), photomicrographs of all
the serial sections were captured. We used the virtual slice feature of Neurolucida that
allowed us to capture the images at 4× magnification in smaller blocks and finally merge
them to yield a holistic image of an entire section. In digital format, the images can be
zoomed in without losing much of the detail. The images were post processed in Adobe
Photoshop to clear up the background. No changes were made to the actual photomicro-
graphic images captured.

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 Introduction xvii

Labeling
The images were labeled based on the nomenclature used by Paxinos, with some
modifications to suit the need of the atlas. For the P-1 rat brain, we labeled most struc-
tures using The Atlas of the Developing Rat Nervous System (Paxinos et al., 1994).
Because the P-7 and P-14 brain structures are similar to those of adult rat brains, we
followed the nomenclature of The Rat Brain in Stereotaxic Coordinates (Paxinos
et al., 1998).
The labeling of the respective structures was done on the actual photographic images
rather than on the classic line diagrams that most atlases utilize. This allows the user to
correlate the structures and their names easily. We decided not to demarcate the areas on
the brain as the brain is a very plastic structure and the areas could not be distinguished
from each other easily. The major parts of the brain were labeled via Adobe Photoshop
without the inclusion of minor details.
We used cresyl violet stain to identify the structures. This posed limitations on iden-
tifying some of the smaller nuclei that required special staining. Most of the structures
were identified based on the proximity to the surrounding structures and outstanding
landmarks; for example, striatum was identified based on its patchy matrix and close
proximity to the corpus callosum.
The most difficult structures to identify were the thalamic nuclei in the P-1 brains. We
restricted our labeling to major nuclei only to avoid confusion. Since the brain develops
very quickly in the first few days after birth, it is very difficult to demarcate the develop-
ing nuclei without in-depth knowledge of that area of research. The other structure that
posed certain limitations in labeling was the cerebellum of the P-1 brain. The lobules are
very different from those of the adult brain.
We tried to keep most of the sagittal brain sections intact. However, we encountered a
few sections in which the cerebellum could not be kept intact with the rest of the brain.
The distances between adjacent plates of the sagittal sections were estimated from the
midline using the section thickness; the midline was considered the absolute zero.
This photographic atlas can assist neuroscientists and students to identify and understand
the developing rat brain structure. However, it does not cover stereotaxic coordinates.

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 Abbreviations

Numbers 1 through 10 Cerebellar lobules

aca Anterior commissure
Acb Accumbens nucleus
AcbC Accumbens nucleus core
AcbSh Accumbens nucleus shell
AICtx Agranular insular cortex
Alv Alveus of hippocampus
AM Anteromedial thalamus
amy Amygdala
Amy Amygdaloid nuclei
AICtx Agranular insular cortex
AO Anterior olfactory bulb
APit Anterior pituitary gland
apons Anterior pons
APT Anterorpretectal nuclei
AT Anterior thalamus
AuCtx Auditory cortex
AV Anteroventral thalamus
Bamy Basal amygdala
BLA Basolateral amygdala
CA1 CA1 field of hippocampus
CA2 CA2 field of hippocampus
CA3 CA3 field of hippocampus
cc Corpus callosum
Cer Cerebellar peduncle
cer Cerebellum
Cernu Cerebellar nuclei
CG Central gray matter
CgCtx Cingulate cortex
CM Central medial thalamic nuclei
Cop Copula of pyramis
Cp Caudate peduncle
CPu Caudate putamen
Crus1 Crus 1 of ansiform lobule
Crus2 Crus 2 of ansiform lobule
Ctx Cortex
Cu Cuneate nucleus
D3V Dorsal third ventricle
DG Dentate gyrus
Dhippo Dorsal hippocampal formation
DLG Dorsolateral geniculate nuclei
DR Dorsal raphe nuclei
DTg Dorsal tegmental nuclei
E Ependyma and subependymal layer
Ec External capsule
Ent Entorhinal cortex
EPI External plexiform layer, olfactory bulb

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