Download The Existence of a Layer IV in the Rat Motor Cortex

The Existence of a Layer IV in the Rat
Motor Cortex
T. S. Skoglund, R. Pascher and C.-H. Berthold
We have reconstructed the laminar pattern of rat primary motor
cortex (Fr1) using a computerized analysis system based on the
so-called ‘optical dissector’. Data were visualized on a graphics
terminal. In contrast to current views, which state that there is no
prominent layer IV in the motor cortex of the rat, our method of
analysis revealed a genuine layer IV consisting of densely packed
small neurons.
We used perfusion fixed cerebral cortex (5% glutaraldehyde)
from seven adult male Sprague–Dawley rats (400–500 g). The
brains were removed, kept in phosphate buffer and divided by a
median cut. Each brain was glued with Histoacryl® (Braun,
Melsungen, Germany) to a support and the cortical area Fr1
(Zilles, 1985) (primary motor cortex; Fig. 1) was sectioned from
its lateral side in sagital sections (six brains) or from its anterior
pole in coronal sections (one brain) in a consecutive series of
50-µm-thick sections using an Oxford Vibratome®. Three to four
sections from each series were used for the counting. In brief,
the staining and the counting methods were as follows (see
Skoglund et al., 1997): the sections were stained in Richardson’s
solution (Richardson et al., 1960) and mounted in 90% glycerol
in 10% PBS on glass slides. This procedure avoids shrinkage and
highlights the neurons (Skoglund et al., 1997). A computersupported system was used to digitize the sections and to count
and measure the neurons. The system consisted of a CCD camera
(DAGE-MTI CCD72E) mounted on a light microscope (Leitz
Aristoplan) equipped with a motorized object stage (Positioniersystem MCL, Märzhäuser Wetzlar, Germany). The three axes, x, y
and z, of the motorized object stage were controlled by a
computer (Toshiba T1000). The CCD camera was connected to
a Silicon Graphics Indy workstation equipped with a 24-bit Indy
Video card. The reconstructions of the distribution of the
neurons were visualized on a Megatek 9300 graphic terminal
(Skoglund et al., 1993).
Images were digitized at a number of sequential positions
from the pial surface down to the white matter along a vector
parallel to radial elements of the cortex (i.e. to neuronal
microcolumns, dendritic bundles and blood vessels). At each
position along the vector, seven optical sections were digitized,
each with a width of 200 µm and a distance of 5 µm between
their focal planes. A compound image, referred to as a cortical
image, reaching from the pial surface down to the white matter
was constructed for each of the seven optical sections. Together
the seven cortical images formed what we will refer to as a ‘3-D
mosaic’, measuring 200 µm × 30 µm × cortical thickness. Our
program ‘Mark’ was then used to count the number of neurons
in a counting frame (i.e. counting box) applied within the ‘3-D
mosaic’. The counting frame measured 170 µm × 25 µm ×
cortical thickness. ‘Mark’ is a program developed by us and
based on the optical dissector (Gundersen, 1986), which is an
unbiased method not dependent on assumptions of cell size and
cell shape. The technique means, in practice, that the neurons
are counted when they first come into focus as one focuses
through a known distance of a thick section. Since large neurons
have a greater probability of being counted, cells that are in
focus in the first focal plane are excluded. Neurons crossing the
left side or the bottom of the counting frame are also excluded.
The position of each neuron was stored in a database.
The organization of neuronal cell bodies in six layers parallel to
the pial surface is one of the most conspicuous features of the
mature mammalian neocortex. The lamination is due to
variations in the packing density and the shapes and sizes of the
neurons as traced from the pial surface down to the white
matter. The first systematic descriptions of the cortical layering
were provided by Meynert (Meynert, 1867, 1869, 1872). The
concept of a six-layered neocortex developed as a result of
Brodmann’s studies (Brodmann, 1903, 1909, 1912; Jones, 1984;
Kemper and Galaburda, 1984). An exception to the six-layer
pattern was found in the adult primate motor cortex, which
Lewis (Lewis, 1878; Kemper and Galaburda, 1984) described as
being five-layered, with the conventional fourth layer missing. A
similarly five-layered motor cortex has also been described as
relevant for the rat and for the mouse, and is, according to the
current literature, accepted as the norm for these species as well
(Donoghue and Wise, 1982; Zilles, 1985; Beaulieu, 1993; Zilles
and Wree, 1995). However, in an often overlooked paper, Krieg
(Krieg, 1946) describes the motor cortex of the rat as being
six-layered including a conventional lamina IV. Because the rat is
one of the most commonly used species in neuroscience, the
confusion with regard to whether its motor cortex contains or
lacks a conventional lamina IV should receive some further
illumination.
In order to study the cytoarchitecture of the cerebral cortex
and to examine, for example, the cortical lamination, we have
developed a computer-based system for the reconstruction of
neuronal organization (Skoglund et al., 1997). The system
creates a database which includes the coordinates and sizes of
neurons, and these can be visualized either by displaying the
neurons as spheres or by plotting the neuronal distribution as a
function of the distance from the pial surface. In this way,
laminar borders are detected as changes in the packing density
of the neurons — a method used earlier to detect both neuronal
(Ryzen, 1956) and nerve fiber stratification (Sanides, 1972;
Wagner et al., 1986).
When we used our system to study rat neocortex we found the
distribution of neurons in the motor cortex inconsistent with the
current view of five layers but in line with Krieg’s findings
(Krieg, 1946) of a six-layered pattern. This paper presents our
results indicating the existence of a layer IV in the motor cortex
of the adult rat.
Institute of Anatomy and Cell Biology and MEDNET-laboratory,
University of Göteborg, Medicinaregatan 3, S-413 90 Göteborg,
Sweden
Cerebral Cortex Mar 1997;7:178–180; 1047–3211/97/$4.00
Figure 1. Micrograph from a sagital section stained in Richardson’s solution. Area Fr1
(primary motor cortex) used for counting.
When the sections were examined under light microscope
without computer assistance, the motor cortex appeared
five-layered, i.e. lacking layer IV. A computer-assisted reconstruction of the distribution of neurons was then made by
displaying the position of each neuron as a sphere in a prism of
the cortex measuring 170 µm × 75 µm × cortical thickness (Fig.
2b). With this approach, a distinct increase in the density of
neurons became apparent at the bottom of layer III, ∼600 µm
from the pial surface (Fig. 2b). The increase became even more
evident when the distribution of the neurons was plotted versus
the distance from the pial surface. As shown in Figure 2c, layer I
contains few neurons, whereas layer II is very cell rich. The
neuronal density falls when entering layer III but increases again
before entering the relatively low density layer V. The location of
the borders was obtained by derivating the density data. In the
derived density data the maximum changes could be found, thus
indicating a lamina border.
The size (diameter) of the neuronal perikarya was measured
during the counting procedure and the volume was calculated by
assuming that the cell bodies were spherical. The mean volumes
of the neuronal somas along an axis from the pial surface to the
white matter are plotted in Figure 2d. A decrease in the variable
is seen at the bottom of layer III. The same pattern was detected
in all investigated animals. Layer IV is usually described as
containing densely packed neurons of small size (Braak, 1980).
This description matches the neurons in the layer we found at
∼600–800 µm from the pial surface. Here, the density is higher
than in layers III and V (Fig. 2c), and the neurons are relatively
small (Fig. 2d). We interpret this layer in the adult rat motor
cortex as being a genuine layer IV, and our results thus support
the findings of Krieg (1946).
Differences in the fundamental organization between the
mature neocortex of the rat and that of more advanced species
have been described earlier (Krieg, 1963; Wagner and Wolff,
1982; Marín-Padilla, 1992) and have been interpreted as being
part of the evolution towards increasingly more ‘complicated’
brains (Innocenti and Kaas, 1995; Northcutt and Kaas, 1995).
However, studies of fetal monkey (Huntley and Jones, 1991) and
human (Ramón y Cajal, 1900; Brodmann, 1903; Marín-Padilla,
1970) brains have shown a six-layered motor cortex. In the
postnatal stage there is an increase in the size of large pyramidal
neurons of the relatively low-density layers III and V and an
increase in the degree to which they become intermingled at the
expense of layer IV. This leads to a progressive reduction in
Figure 2. (a) Micrograph and (b, c and d) reconstructions of area Fr1. (a) Micrograph
from 50-µm-thick section of Fr1 stained in Richardson’s solution. (b) Reconstruction of
the positions of the neurons within a cortical prism from Fr1. Each neuron is shown as
a sphere (all spheres are of the same size). A layer IV can be detected ∼600–800 µm
from the pial surface. (c) Linear plot showing the relative distribution of neurons versus
the distance from the pial surface. The high density region 600–800 µm from the pial
surface is what we refer to as layer IV. (d) Linear plot showing the medium size (volume)
of the neurons versus the distance from the pial surface. The layer IV is here
recognizable as a decrease in the mean size of the neurons.
neuronal packing density and finally to an apparent obliteration
of layer IV. We do not know whether our computer-assisted
method would resolve a fourth layer in adult monkey and human
motor cortex when sorting out the neurons according to size.
In conclusion, we have confirmed the presence of a genuine
though somewhat elusive lamina IV in the adult rat motor
cortex. According to our results, the mature rat motor cortex is
subdivided into the conventional six layers of the following
approximate thicknesses as estimated for lamina I through
lamina VI: 210, 230, 180, 190, 520 and 550 µm respectively. We
would like to emphasize the importance of recognizing the
presence of a lamina IV in forthcoming descriptions of the
mature rat motor cortex.
Notes
This work was supported by the Medical Faculty in Göteborg, by the
Swedish MRC proj. no. 3157, by the Göteborg Medical Society, by
Lundberg’s Research Foundation, by Anna Ahrenberg’s Foundation and
by Rådman Ernst Colliander’s Foundation. We thank Marieanne Eriksson
Cerebral Cortex Mar 1997, V 7 N 2 179
and Rita Grandér for excellent technical assistance. The facilities of the
MEDNET laboratory were used.
Address correspondence to Dr Thomas S. Skoglund, Institute of
Anatomy and Cell Biology, University of Göteborg, Medicinaregatan 3,
S-413 90 Göteborg, Sweden.
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