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Methods
Surgery |
Histology and Tissue Shrinkage |
Illustrations |
Brain Size |
Delineation of Structures
Surgery
Adult animals were deeply anaesthetised with 5% thiopentone
sodium (“Pentothal”, Boeringer Ingelheim Pty.
Ltd., Artarmon, N.S.W.; 0.5-1.0 ml, as needed) and were then
placed in a stereotaxic device so that the skull was tilted
12 o down with respect to an imaginary line bisecting the
lachrymal foramina and extending to the centre of the au
ditory foramen (see Fig. 1). The precise stereotaxic co-ordinates
of Bregma were recorded. A series of craniotomies were made
in the dorsal surface of the skull and stainless steel needles
inserted into the brain perpendicular to the imaginary line.
The needles were placed at pre-determined locations with
respect to Bregma and their stereotaxic co-ordinates recorded.
Fig. 2 is an X-ray illustration showing location of the needles
(in this case horizontally-placed needles are also present).
Following surgery, a lethal dose of “Pentothal” was
administered and the animal perfused transcardially with
normal saline and 10% formalin. After decapitation, the skull
was immersed in formalin for a week. It was then repositioned
in the stereotaxic apparatus, a large plate of bone removed
and, using a scalpel blade set perpendicular to the imaginary
line between the lachrymal and auditory foramina, the brain
was transected coronally at Bregma. The brain was carefully
removed from the skull and the stainless steel withdrawn.
It was then placed in 30% 0.1M sucrose buffer where it remained
for a month. Fig. 3 comprises photographs of a perfused brain
from three perspectives: lateral, dorsal and ventral.
Histology and Tissue Shrinkage
For an anatomical atlas to be of practical use to researchers,
the stereotaxic co-ordinates of its various structures must
accurately reflect those of the brain of the living animal.
During fixation and staining processes, however, significant
tissue shrinkage is known to occur. For example, Braitenberg
and Schüz in Anatomy of the Cortex (1991) indicate
that with Nissl staining of frozen sections, the tissue may
shrink to as much 68% of its original volume. In preparing
the present atlas, allowance was made for tissue shrinkage
so that illustrations accurately represent the living brain.
Brains upon which the atlas was based were set in either
celloidin or gelatine-albumin blocks and a series of sequential
frozen coronal sections was cut on a Leitz Wetzlar sledge
microtome. The thickness of the sections was either 40 or
60 microns, depending on the individual experiment; needle
tracks were clearly identifiable in relevant sections. The
visibility of these tracks made it a relatively simple matter
to calculate their spatial separations. These distances were
then compared to those separating the needles which had been
placed in the brains of anaesthetised animals prior to perfusion.
The resulting tissue shrinkage factor was subsequently calculated
at 16%.
Cut sections were collected in phosphate buffer and mounted
on gelatinised slides. After air drying these were stained
with Cresyl violet and Fast blue in order to visualise the
cells Nissl substance and fibres. Slides were then cover-slipped.
Illustrations
The sections illustrated and their matching drawings are
separated by a distance of 1 mm in the rostral-caudal plane
and are referenced to Bregma (the junction of the coronal
and sagittal sutures).
Sections were scanned into Adobe Photoshop using an Epson
Perfection 4870 scanner. A Bausch and Lomb vertical projector
was used to display large images of the sections on paper
and drawings of one half of the brain designating major features
were prepared. Artistic licence was taken only when part
of a section was missing or severely distorted, and then
only after careful consideration of adjacent and contralateral
sections. Tissue damage caused by needle tracks was not erased.
Consequently, the illustrations accurately reflect the sections.
The drawings were subsequently photo-reduced before being
scanned with an AVR Scanner into a Macintosh computer using
Adobe Photoshop 2 software. Dorsal-ventral and medial-lateral
stereotaxic co-ordinates are ascertainable by reference to
a 1 mm grid superimposed on the drawings. The grid’s
medial-lateral zero point commences at the brain’s
midline, while its dorsal-ventral zero is positioned at the
surface of the cortex.
Abbreviations shown on each drawing and the structures
to which they refer are listed below each section and its
drawing.
Three series of sections were used in compiling the atlas.
Brain Size
In Fig. 4 lateral and dorsal photographs of the brain were
superimposed on X-ray images of the skull in order to indicate
the position of the brain in situ
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