Results 2. Coronal organization Directory UMM :Data Elmu:jurnal:B:Brain Research:Vol881.Issue1.2000:
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played. The cross sections of blood vessels were used as assessed by several means. Sulcal patterns were used to
landmarks to align multiple sections. Figs. 4–6 consists of establish the border between areas 19 and 21a [21,54,55]
data from multiple tissue sections that were collapsed onto and alternate tissue sections stained for CO were used to
a single plane. identify the border between areas 18 and 19, as CO
For every image, the position of each charted cell was staining in area 19 is lighter than that observed for areas 17
overlaid with a 2D Gaussian kernel with a radius of 0.25 and 18 Fig. 1 [3]. The area 18 19 border established the
mm. From the resulting image, transects were drawn location of area 19. Area 21a was identified by the fact it
across the bands and labeling density of the bands was adjoins area 19 at the posterior crown of the suprasylvian
calculated Fig. 5. The peaks in the profile plots, gener- gyrus Fig. 1 [55]. As corresponding visuotopic areas are
ated from the transects perpendicular to the bands, were commonly connected [13,44], injections of retrograde
measured to give an estimate of band periodicity Fig. 5. tracer in area 21a, which has a representation limited to the
The periodicity measurements were then averaged for an central 208 of visual field [55], would be expected to label
overall mean periodicity. For every band, five transects regions of central representation in other areas. Cell
were also taken to measure band width Fig. 6. Given labeling from 21a injections was confined to the region of
some variability in band shape, the five measurements per central representation in areas 17 and 18. Also, within area
band which were averaged for each band were deemed 19 labeling was found in the region of central representa-
necessary to give a true estimate. The mean values for each tion, which is located in the posterior half of this area [54].
band was then averaged for an overall estimate of band Furthermore, an extension of the 21a injection into the
width. Transects were also taken along the mediolateral neighboring lateral suprasylvian LS area would result in
axis of the bands to measure changes in labeling density labeling of the peripheral representation in other areas,
Fig. 6. Measurements were not adjusted to take into since the LS region adjacent to 21a contains peripheral
account shrinkage of tissue during fixation and processing representation [33,37,55]. However, there is no label found
and are thus minimal estimates of width and periodicity of in the peripheral representation regions of areas 17, 18 and
the bands. 19, further confirming the injection was restricted to area
21a.
3. Results 3.2. Coronal organization
3.1. Injection placement From the coronal experiment, it is evident that the
neurons in area 19 that project to area 21a are located in Correct placement of injections into area 21a was
columnar register Fig. 2; large arrows extending through
Fig. 1. A tangential section of flattened cortex stained for CO. The area 18 19 border, marked by the dashed line with white asterisk, is identified by the edge of a dark CO oval, which encompasses areas 17 and 18. In this section, the genu at the posterior end of the suprasylvian sulcus is darkly stained by
CO and is marked by the white arrow. Above the genu the multiple pipette tracts in area 21a are visible. The thin solid lines correspond to the lateral sulcus Lat and the suprasylvian sulcus SS. A, anterior; L, lateral; LS, lateral suprasylvian area.
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Fig. 2. A photograph of a coronal slice with retrogradely labeled cells in area 19 following an injection of WGA–HRP in area 21a. This section has been immunohistologically counterstained with SMI-32, which labels the nonphosphorylated forms of neurofilament protein. SMI-32 immunoreactivity labels
somata and apical dendrites [2] and appears lighter than the dense TMB reaction product of WGA–HRP labeled cells. The WGA–HRP labeled cells in area 19 are organized in patchy columns, two of which are shown by the large arrows above the pial surface. The small arrows indicate examples of individual
WGA–HRP labeled cells. While layers 2 3 contain the most labeled cells, layers 4 and 5 demonstrate modest labeling and layer 6 has very few labeled cells. Layer 1 does not contain any labeled cells. Scale bar5500 mm.
layers 2 to 6 Fig. 2; small arrows. The number of patches from area 19 to area 21a is organized into discrete,
of labeled cells per tissue section range from 0 to 4 and irregular bands. In most cases, these bands are elongated
have a centre-to-centre spacing ranging from 0.6 to 0.9 roughly in the mediolateral direction Figs. 3, 4, but there
mm. The majority of labeled cells within the columns are is some variation in the mediolateral orientation of the
found in layers 2 3. Layer 4 and 5 contain moderate bands Fig. 5A. The bands are found in the posterior half
amounts of labeling while labeling in layer 6 is scarce. In of area 19 and range in number from 2 to 6 Table 1.
some sections, a sparse number of lightly labeled cells are Variation in band number may be due to differences in the
found outside the columns in layers 2 3 and 5. size of the effective injection site in area 21a. It may also
be a result of the complexity of some of the bands which form bridges arrows in Fig. 6A. The bridges between the
3.3. Tangential organization bands may actually represent a separate patchy system
similar to the one seen in the coronal cases. Although the Retrograde labeling of cells reveal that the projection
density of labeled cells within bands sometimes fluctuates,
T .H. Stewart et al. Brain Research 881 2000 47 –56
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Fig. 3. A dark-field photograph of area 19 of WGA–HRP labeled cells after an injection into area 21a. The two elongated 21a efferent bands in area 19 are marked by white arrowheads. Scale bar50.5 mm.
the presence of labeling between regions of high density, creates a visibly discrete band.
3.4. Periodicity of tangential bands A measurement of the distance between density peaks,
which was determined by density profiles taken from line transects, was used to calculate the mean periodicity Fig.
5C. The mean periodicity of the area 19 tangential bands is 2.6 mm. The S.E. is relatively high at 0.24, indicating
that considerable variation exists. Despite this irregularity in spacing, in no case do the separate bands merge to
create uniform labeling across area 19. Area 19 clearly demonstrates fluctuations in labeling density in which
areas of high density are separated by areas of diminutive density.
Fig. 4. A chart of labeled cells following an injection of CTX–Au in area
3.5. Width and mediolateral density of tangential bands
21a. Largely spaced bands of label are visible in area 19 whose borders are shown by dashed lines. A thin solid line marks a tear in the tissue.
The width and mediolateral density of the bands are also
Note the clusters of labeled cells just medial to the area 18 19 border. A, anterior; L, lateral. Scale bar52 mm.
assessed using procedures similar to those used to measure
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Table 1 The breakdown of measurements from separate cases of area 19 efferent
a
bands Animal
No. of No. of
Mean Mean width
Fig. number
charted bands
periodicity mm
no. sections
mm T3
4 4
3.56 0.98
3 T7
2 3
2.70 0.73
– T14
2 5
3.25 1.03
1 T17
3 6
1.76 0.79
5 T18
3 4
2.48 0.95
6 JB1
3 2
1.49 0.64
4
a
The first column contains the tracking number of each animal. The second column represents the number of alternate sections that were
stained for retrograde labeling and charted to create a single graphical image used for the analysis. The number of area 19 efferent bands found
in each animal varies between two and six, as can be seen from column three. The next two columns represent the mean periodicity and width
values for the bands found in each case. The figure number corresponding to each individual case is found in the last column.
after multiple sections are charted, aligned and collapsed onto one plane it is evident that there are patches of
increased staining within the band, occurring on average at 0.9 mm intervals. This patchiness is visible in individual
sections and may be related to the columns of cell clusters in the coronal plane Fig. 2. However, clusters of dense
areas within the bands are not segregated into individual units, as seen in many of coronal cases. Rather, within the
bands there appears to be a low level of continuous labeling. This suggest the possibility of a periodic sub-
band structure.
The density of the band along the width or anterior posterior axis is very consistent. Fig. 6D shows typical
transects through the width of the band. The relatively normal distribution of density along this axis is found in all
animals. This consistency suggests that there is no sub- structure or clustering in the anteroposterior direction. The
only changes in cell labeling density that is encountered in this direction is the area 19 bands which are distinct from
each other.
Fig. 5. An example of a cell chart used to measure periodicity of the area 19 efferent bands. A A cell chart confined to labeled cells in area 19
after an injection of CTX–Au in 21a. The clusters of labeled cells are approximately perpendicular to the area 18 19 border and are elongated
3.6. Tangential organization of area 18
along the mediolateral axis. Variation in the orientation of the bands is common between animals. B The same cell chart overlaid with a 2D
In the process of examining area 19 it was noticed that
Gaussian kernel. A transect dashed line has been taken through five bands. C The periodicity of the bands was measured by measuring the
area 18 also consistently demonstrated clusters of labeled
distance between peaks in the transect. A, anterior; L, lateral. Scale
neurons on the medial side of the posterior half of the area
bar52 mm.
18 19 border. These clusters of cells are evident in all tangential cases. As seen in Fig. 4 these clusters are
elongated mediolaterally and have an approximate spacing periodicity. Examples of the profile plots used to measure
of 0.9 mm. The spacing of these area 18 clusters is similar the mediolateral density and width of each band are shown
to the spacing of patches of labeling in area 17 0.6–0.9 in Fig. 6C and D. The average band width is 0.9 mm
mm that appear after a retrograde tracer injection in area S.E.50.03.
21a. These patches of label in area 17 are known to The profile plots reveal variation in density along the
colocalize with CO blobs and are a characteristic feature of mediolateral, axis of the bands Fig. 6C. Furthermore,
the striate projections to area 21a [8].
T .H. Stewart et al. Brain Research 881 2000 47 –56
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Fig. 6. An example of cell charts used to measure mediolateral density and width of the area 19 efferent bands. A A cell chart of the retrograde labeling in area 19 after injections in area 21a. Notice the branching of some of the bands small arrows. B The same cell chart overlaid with a 2D Gaussian
kernel. Three transects along the mediolateral axis of a band are illustrated 1, 2 and 3. Three transects along the width of the bands are illustrated A, B and C. C The profile plots generated by transects 1, 2 and 3 in Fig. 6B. Note the uneven density along the transect. This was a common feature found in
all cases. D The profile plots generated by transects A, B and C in Fig. 6B. The density of labeled cells along the transect peaks in a smooth Gaussian fashion. The consistency of density along the anterior posterior axis was found in all cases. A, anterior; L, lateral. Scale bar52 mm.