48 T
instance, most neurons in area 21a exhibit high spatial mg kg, Austin Laboratories were given to the animals to
frequency selectivity and are orientation, but not motion, reduce brain edema.
selective [14,31,53]. This study addresses area 21a inputs arising from area 19, as area 19 is a pivotal visual area
2.2. Histology with connections to over 11 extrastriate visual areas
[4,18,49], suggesting that it plays a role in several process- The animals were given an overdose of barbiturate
ing streams in the cat visual system. anesthetic three days after surgery. They were then per-
In this study we made large injections of a retrograde fused transcardially with 700 ml of phosphate buffer 0.1
tracer to saturate area 21a, as identified stereotaxically. The M, pH 7.2 with 0.5 sodium nitrite. In six cases, a
organization of the efferent projection to area 21a was solution of 2 paraformaldehyde in phosphate buffer was
examined in both the tangential and coronal planes. delivered with a perfusion pump at a rate of 60 ml min for
Retrograde labeling in area 19 revealed a complex pattern 4 min. The visual cortex was then unfolded and flattened
of bands in area 19, elongated in the mediolateral direc- tangentially as described by Olavarria and Van Sluyters
tion. Within these bands, clusters of dense staining was [36]. The visual hemispheres were left between two glass
found, which suggests a substructure to the overall banded slides submersed in 4 paraformaldehyde and 20 suc-
pattern. rose in phosphate buffer. After 5 h, the glass slides were
removed and the sections were allowed to free float for 8 h. The tissue was then cut tangentially at 50 mm on a
2. Materials and methods microtome. In one case with WGA–HRP injected, 4
paraformaldehyde was used in the perfusion and after- 2.1. Animals and surgical procedure
wards the visual cortex was cryoprotected and 50 mm coronal sections were cut on a microtome.
Seven normal adult cats of both sexes were used. Five of Alternate tangential sections were stained for CO using
the animals received injections of cholera toxin subunit B a cobalt and nickel enhancement method [9,15,26]. The
conjugated to 7 nm colloidal gold CTX–Au; List Bio- CO staining solution contained 20 mg diaminobenzidine,
logical; 1 in 0.9 sterile saline. Two animals were 30 mg cytochrome C, 15 mg catalase and 2 g sucrose in 50
injected with wheat germ agglutinin–horseradish peroxi- ml 0.01 M phosphate buffer pH 7.2 to which 5 ml of 1
dase WGA–HRP; Sigma; 1 solution in 0.9 saline. nickel ammonium sulphate was added. One percent cobalt
The animals were pre-anesthetized with a subcutaneous chloride was then added dropwise until the solution
injection of glycopyrilate 0.05 mg kg, Associated Vet- became cloudy. The tissue sections were incubated in the
erinary Purchasing, Co. Ltd., followed by an intramuscu- solution for 4–6 h.
lar injection of ketamine 20 mg kg, Associated Veterinary The remaining tissue sections from these six cases were
Purchasing, Co. Ltd. and diazepam 2 mg kg, Sabex Inc.. stained for retrograde labeling. For five cases, CTX–Au
Animals were also given an intramuscular injection of was visualized by silver intensification. This was done by
dexamethasone 0.5 mg kg, Austin Laboratories to pre- incubating the sections for 1–2 h in a silver enhancement
vent brain edema. During surgery anesthesia was main- solution Jannsen IntenSEM. The remaining tangential
tained by inhalation of trifluourethane halothane, M.T.C. case plus the one coronal case were reacted for WGA–
Pharmaceuticals. Under sterile surgical conditions an HRP using the standard tetramethylbenzidine TMB
incision was made along the midline of the head and a method [30]. This was followed by a stabilization in
long-lasting local anesthetic, 0.25 bupivacaine hydro- chilled
ammonium heptamolybdate
and cobalt–
chloride Marcaine, Winthrop Laboratories, was injected diaminobenzidine [22]. Half of the coronal sections were
into the incision. A craniotomy and duratomy were per- then processed for SMI-32 immunoreactivity using a
formed at stereotaxic co-ordinates AP 27 to 12 and ML monoclonal antibody Sternberger Monoclonals Inc..
17 to 115 mm to reveal area 21a. SMI-32
immunoreactivity helped
identify laminar
Tracer injections were made just medial to the genu boundaries seen in Fig. 2.
along the posterior portion of the suprasylvian gyrus. Long micropipettes, with tapered ends that had inner diameters
2.3. Data analysis of 16–20 mm, were used for injections. The pipettes were
placed perpendicular to the surface of area 21a and The labeled cells were charted as described by Boyd and
injections were made at a cortical depth of 500 mm with a Matsubara [3]. Briefly, custom software was run on a PC
spacing of approximately 500 mm. The injections covered computer. A Nikon Optiphot compound microscope with
a 535 mm area and a total of 7–8 ml of tracer was an x –y coordinate stage encoder and a drawing tube was
injected. These multiple injections created a large aggre- used to determine the spatial layout of labeled cells and
gate injection that saturated a large portion of area 21a landmarks in each tissue section. The x and y coordinates
with tracer. Gelfoam was then put in the craniotomies and of the labeled cells and radially penetrating blood vessels
the fascia and skin along the incision site sutured. Follow- were recorded and transferred into IGOR Professional 3.1
ing the surgery, daily injections of dexamethasone 0.25 software Wavemetrics, Inc. and were graphically dis-
T .H. Stewart et al. Brain Research 881 2000 47 –56
49
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
