Cerebral Cortex, Vol. 12, No. 7, 671-691,
July 2002
© 2002 Oxford University Press
Human-specific Organization of Primary Visual Cortex: Alternating Compartments of Dense Cat-301 and Calbindin Immunoreactivity in Layer 4A
University of Louisiana at Lafayette, Cognitive Evolution Group, New Iberia, LA 70560, USA
Todd M. Preuss, Ph.D., University of Louisiana at Lafayette, Cognitive Evolution Group, 4401 West Admiral Doyle Drive, New Iberia, LA 70560, USA. Email: tmpreuss{at}louisiana.edu.
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Abstract |
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There is evidence that the cortical anatomy of the magnocellular (M) visual pathway, which carries information about motion and luminance contrast, was modified in human evolution. Recent results indicate that layer 4A of humans contains a meshwork of tissue bands that stain densely for nonphosphorylated neurofilament (NPNF), a protein that is preferentially expressed in elements of the M pathway, whereas apes and monkeys lack a comparable pattern. Here we examined the distribution of staining for Cat-301 – a monoclonal antibody well established to stain M-related structures preferentially – in area V1 of humans, apes (chimpanzees, orangutan), Old World monkeys (macaques) and New World monkeys (spider monkeys, squirrel monkeys). Single-staining experiments, using a peroxidase–tetramethylbenzidine (TMB) reaction, revealed alternating zones of dark and light staining for Cat-301 in layer 4A of humans, similar to those observed with NPNF. Double-staining studies in humans revealed that Cat-301-immunoreactive somas and neuropil were localized within the same tissue bands that stained strongly for NPNF and, furthermore, that these bands alternated with irregularly shaped territories that stained very strongly for calbindin. Nonhuman primates, by contrast to humans, displayed weak Cat-301 and calbindin staining in layer 4A. The co-localization of Cat-301 and NPNF in human layer 4A, and the weak staining for these molecules in layer 4A of other primates, suggests that the cortical representation of the M channel was modified in recent human evolution. The calbindin-rich compartments in human layer 4A cannot be related to a particular geniculostriate pathway on neurochemical grounds; they may constitute an interneuronal population that increased in human evolution.
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Introduction |
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Comparative studies of the mammalian primary visual area (V1; area 17; striate cortex) have revealed some notable phyletic variations in cellular, laminar, compartmental and connectional organization. Within the primate order, differences have been documented between prosimians (lemurs, lorises) and anthropoids (monkeys, apes and humans), and between anthropoid groups (Hässler, 1967
; Casagrande and Kaas, 1994; Preuss, 1995). There is now evidence that the organization of area V1 in humans differs in certain aspects from that of most other primates, particularly in the organization of layer 4A. In most New World and Old World monkey species that have been examined, layer 4A of the primary visual area is marked by a thin, dense band of cytochrome oxidase (CO) staining that is coincident with a thin layer of terminals arising from the parvocellular layers of the lateral geniculate (Horton, 1984; Wong-Riley, 1994). This sheet is punctuated by columns of apical dendrites and short stacks or cones of pyramidal cell bodies extending upward from layer 4B into 4A (Peters and Sethares, 1991; Hendry and Bhandari, 1992; Peters, 1994). These punctuations give the sheet of CO staining and geniculate terminals a distinctively lattice-like or ‘honeycomb’ appearance in sections cut parallel to the pial surface (Hendrickson et al., 1978). Humans, however, lack a CO-dense band (Horton and Hedley-Whyte, 1984), as do chimpanzees, the animals most closely related to humans (Preuss et al., 1999). The reduction of the CO-dense layer 4A band in the evolution of the hominoid primates (i.e. apes and humans) suggests that there may have been a corresponding reduction of the parvocellular geniculate projection to this layer (Horton and Hedley-Whyte, 1984; Preuss et al., 1999).
Layer 4A apparently underwent further modification during recent human evolution. Preuss et al. (Preuss et al., 1999) stained primary visual cortex from humans and a variety of other nonhuman primate species (including apes, Old World monkeys, and New World monkeys) for nonphosphorylated neurofilaments (NPNF) using monoclonal antibody SMI-32. In layer 4A of humans, they observed a distinctive pattern of NPNF staining in which bands of darkly stained tissue, consisting of dense neuropil with embedded cell bodies, were intermingled with lightly stained territories, giving the layer a mesh-like appearance in coronal sections. In apes and monkeys, by contrast, layer 4A lacked prominent bands of tissue stained for NPNF; instead, layer 4A was generally lightly stained in these species, and most of the stained elements that were observed consisted of apical dendrites of pyramidal cells with somas located in deeper layers. A comparable pattern of differences between humans and nonhuman primates was obtained using an antibody against microtubule-associated protein 2 (MAP 2), which also stains pyramidal cell bodies and dendrites in nonhuman primates, although staining was not as consistent across individuals as with the NPNF antibody. There are other indications that human layer 4A has an unusual pattern of compartmental organization: Hendry and Carder (Hendry and Carder, 1993) reported that human layer 4A exhibited dense but irregular staining for a calcium-binding protein, calbindin D-28k, whereas this layer was more lightly and uniformly stained in the Old World and New World monkeys they examined.
These results raise several important questions about the organization of layer 4A in human primary visual cortex. First, what is the relationship of the different compartments of human layer 4A to the three recognized channels of visual information processing, that is, the magnocellular (M), parvocellular (P) and koniocellular (K) pathways (Livingstone and Hubel, 1988; Casagrande and Kaas, 1994; Callaway, 1998; Hendry and Reid, 2000)? Preuss et al. (Preuss et al., 1999) suggested that the tissue bands that stain darkly for NPNF are related to the M pathway, because NPNF staining is reported to be comparatively dense in the magnocellular layers of the LGN (Gutierrez et al., 1995; Chaudhuri et al., 1996; Vickers, 1997) and in other neuronal populations related to the M pathway (Campbell and Morrison, 1989; Hof and Morrison, 1995; Chaudhuri et al., 1996). There is, however, a molecule that has been even more definitively associated with the M system than NPNF, namely, the antigen labeled by monoclonal antibody Cat-301. Cat-301 stains many different regions of the nervous system, but within the visual system it differentially stains structures related to the M pathway, including the magnocellular LGN layers, layer 4B of area V1, the thick stripes of area V2, and area MT (Hendry et al., 1984, 1988; DeYoe et al., 1990). Cat-301 labels an extracellular-matrix proteoglycan (Zaremba et al., 1989; Fryer et al., 1992; Lander et al., 1997) that is concentrated in the so-called ‘perineuronal nets’ that invest certain classes of neurons (Brückner et al., 1996; Celio et al., 1998).
If the interpretation of layer 4A compartmentation suggested by Preuss et al. (Preuss et al., 1999) is correct, we would expect Cat-301 to yield a mesh-like pattern of staining similar to that seen with NPNF staining, and double-staining studies should demonstrate that Cat-301 is localized within the same tissue bands that stain strongly for NPNF. Weighing against this expectation is a previous study of Cat-301 immunoreactivity in area V1 of humans (Hockfield et al., 1990), which indicated that layer 4A stains lightly in humans, similar to macaques. However, that study was done using diaminobenzidine (DAB) immuno-cytochemistry, which is less sensitive than the tetramethyl-benzidine (TMB) techniques now available (Weinberg and van Eyck, 1991; Llewellyn-Smith et al., 1993).
In the present study, therefore, we revisited the issue of Cat-301 immunoreactivity in area V1 using TMB histochemistry with tissue obtained from humans, apes (chimpanzees and orangutan), Old World macaque monkeys, and New World squirrel monkeys and spider monkeys. We also directly examined the relationship between NPNF and Cat-301 immunoreactivity in human V1 with a sequential double-staining procedure using alternative and readily distinguishable TMB reaction products. In this procedure, the first antibody was localized with TMB stabilized with DAB and cobalt (TMB–DAB–Co), which yields a reddish-brown reaction product, and the second antibody was localized with TMB alone, which yields a blue reaction product. The concept is similar to sequential double-staining techniques using, for example, DAB in combination with benzidine dihydrochloride (Levey et al., 1986), but exploits the greater sensitivity of TMB. This procedure was adopted in preference to a double-immunofluorescence approach, because the high levels of autofluorescing lipofuschin commonly present in human cortex can obscure labeling, and because immunoperoxidase techniques employing TMB are likely to be more sensitive than immunofluorescence and better suited for demonstrating compartmental patterning of neuropil at relatively low magnification. Furthermore, because NPNF is an intracellular protein and the Cat-301 antigen is an extracellular-matrix constituent, it is unlikely that one reaction product would obscure or interfere with the other in any cells that express both molecules.
A second issue investigated here concerns the calbindin-rich territories in human layer 4A reported by Hendry and Carder (Hendry and Carder, 1993). Although Hendry and Carder did not find similar features in macaques and squirrel monkeys, they did not examine apes, so it has yet to be determined whether calbindin-rich territories are a specialization of the ape–human group or a true human specialization. Therefore, we examined calbindin immunoreactivity in apes as well as in humans, macaques, squirrel monkeys and spider monkeys. In addition, we examined in humans the relationship between layer 4A compartments that express calbindin with those that express NPNF or the Cat-301 antigen using the same double-staining technique described above.
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Subjects and Tissue
We examined occipital lobes from a total of nine humans (Homo sapiens), six chimpanzees (Pan troglodytes), one orangutan (Pongo pygmaeus), five macaque monkeys (representing Macaca mulatta, M. nemestrina and M. fascicularis), six squirrel monkeys (Saimiri sciureus), and three spider monkeys (Ateles geoffroyi). Subsets of these cases were used for each of the different immunohistochemical experiments, as described below. The human material was obtained from the brain bank of the Northwestern University Alzheimer's Disease Center (NWADC). The human sample included individuals of both sexes ranging in age from 54 to 78 years of age (median = 70 years). Eight of nine brains were rated as normal control brains on neuropathological criteria by NWADC; one brain showed some evidence of Alzheimer's-related changes. Post-mortem delays ranged from 3 to 23 h (median = 9 h). Five of the chimpanzees, and the one orangutan, came from the University of Louisiana at Lafayette's New Iberia Research Center. One additional chimpanzee came from the Yerkes Primate Center. All had died of natural causes or were euthanized for humane reasons. Post-mortem delays for the apes ranged from 0 to 7 h (median = 4 h). The Old World and New World monkey brains came from animals at the New Iberia Research Center and Vanderbilt University. All were sacrificed with a lethal dose of barbiturate. Procedures involving nonhumans were carried out in accordance with guidelines established by the institutional animal care and use committees of the New Iberia Research Center, Yerkes Primate Center and Vanderbilt University.
The human brain tissue examined in this study came from the posterior-most part of the occipital lobe, extending no more than ~5 cm anterior to the occipital pole. The region of human area V1 examined, therefore, included the central and parafoveal representations of the visual field in area V1 (Horton and Hoyt, 1991), but not the peripheral field representation, located at more anterior levels of the occipital lobe.
The human brains were blocked and fixed by immersion in phosphate-buffered 2–4% paraformaldehyde for 1–4 days. In some cases, larger brain blocks were subdivided in 1–1.5 cm thick slabs prior to immersion to reduce fixation artifact. Three chimpanzee brains and the one orangutan brain were fixed by immersion in aldehydes for 1–4 days. The solutions used for immersion were buffered 2–4% paraformaldehyde solution (two chimpanzees, one orangutan), buffered 4% para-formaldehyde and 0.15% glutaraldehyde (one chimpanzee), and buffered 10% formalin (one chimpanzee). Three additional chimpanzees were fixed by perfusion. One was fixed at the time of death with buffered 4% paraformaldehyde and 0.1% glutaraldehyde, while two others were perfused post-mortem with buffered 2–4% paraformaldehyde. Two macaque brains, removed within 15 min of death, were fixed by immersion in buffered 4% paraformaldehyde. The other Old World and New World monkey brains were fixed by perfusion with buffered 2–4% paraformaldehyde or in a few cases with a mixture of 2–4% para-formaldehyde and 0.08–0.15% glutaraldehyde.
Following fixation, brain blocks were immersed in sucrose or glycerol solutions to prevent freezing artifact, and then stored in an ethylene-glycol-based cryoprotectant solution (Watson et al., 1986) at –20°C. Prior to sectioning, blocks were immersed in buffered 40% sucrose at 4°C for several days to remove cryoprotectant solution. After cutting, tissue sections were stored in cryoprotectant solution at –20°C.
Single Immunocytochemistry: Cat-301
We examined Cat-301 immunoreactivity in five humans, five chimpanzees, one orangutan, four macaques, six squirrel monkeys and two spider monkeys. Cat-301 staining intensity was found to vary across species, so we ran series of dilution and incubation trials for each species. As discussed in the Results, final primary antibody dilutions ranged between 1:4 and 1:40, depending on the species, with incubation conditions ranging from overnight at room temperature to 7 days at 4°C. The Cat-301 antibody was kindly supplied by Dr Susan Hockfield (Yale University).
Sections were prepared for immunocytochemistry by rinsing them in Tris-buffered saline (TBS; pH 7.4) to remove cryoprotectant and immersing them in a series of methanol–hydrogen peroxide solutions to inactivate endogenous peroxidase. Sections then underwent a treatment to digest chondroitin, which partially masks the Cat-301 epitope situated in the core of a chondroitin sulfate-containing proteoglycan (Fryer et al., 1992): sections were rinsed for 10 min in chondroitinase buffer (0.1% bovine serum albumin, 0.1% CHAPS, 0.04% sodium acetate anhydrous, 0.9% sodium chloride, 0.1 M Tris buffer at pH 8) and then incubated in chondroitinase ABC (0.1 unit/ml; Sigma, St Louis, MO, USA) diluted in chondroitinase buffer for 2 h at 37°C, followed by incubation in the same solution overnight at room temperature. After digestion and rinsing, sections were incubated in Cat-301 supernatant diluted in a solution of 5% fetal calf serum, 0.1% Triton, and 0.001–0.1% sodium azide made up in Dulbecco's modified Eagle medium. Following incubation, sections were immersed in biotinylated secondary antibody (12.5 µl/ml; Amersham Pharmacia Biotech, Piscataway, NJ, USA) for 2 h, rinsed, and immersed for 1 h in streptavidin–biotin complex (12.5 µl/ml; Amersham). The sections were then reacted for 8–10 min in TMB solution (0.0025% TMB, 0.05% ammonium paratungstate, 0.1% glucose oxidase, 0.2% d-glucose and 0.004% ammonium chloride), and the reaction product stabilized for 8 min in DAB–cobalt solution (0.1% DAB, 0.02% cobalt chloride, 0.1% glucose oxidase, 0.2% d-glucose and 0.004% ammonium chloride). Preparation of these solutions is described by Llewellyn-Smith et al. (Llewellyn-Smith et al., 1993). Reacted sections were mounted on gelatin-coated slides. Selected sections were counterstained for Nissl with thionin to evaluate the laminar distribution of labeling. In all experiments, control sections were prepared in the same manner as positive sections, except that the primary antibody was omitted from the incubation step. Control sections showed no specific staining.
Single Immunocytochemistry: Calbindin, NPNF and MAP 2
We examined immunoreactivity for calbindin D-28k in six humans, five chimpanzees, one orangutan, three macaques, three squirrel monkeys and three spider monkeys using the TMB–DAB–Co technique. In all species, separate series of sections were stained using a monoclonal antibody, CL 300 (Celio et al., 1990), obtained from Sigma, and using a polyclonal antibody, AB 1778, obtained from Chemicon (Temecula, CA, USA). With both antibodies, staining intensity varied across species, and final dilutions employed ranged from 1:1000 to 1:4000 for the monoclonal antibody and 1:1000 to 1:8000 for the polyclonal. At all dilutions, sections were incubated overnight at room temperature. To visualize the location of labeling using these antibodies, sections were first rinsed in TBS to remove cryoprotectant and immersed in a series of methanol–hydrogen peroxide solutions to inactivate endogenous peroxidase. Sections were then blocked with 3% normal sheep or donkey serum for 1.5–3 h, rinsed in TBS, and incubated in primary antibody under conditions described above. Triton X-100, typically at a concentration of 0.1%, and 0.1% sodium azide were added to the blocking and incubation solutions. Following incubation, sections were immersed in biotinylated secondary antibody followed by streptavidin–biotin complex, and reacted with TMB, DAB and cobalt as described above for Cat-301 immunocytochemistry. Reacted sections were mounted on gelatin-subbed slides, and selected sections were counterstained with thionin. Negative control sections, prepared by omitting primary antibody from the incubation solution, showed no specific staining.
In addition to the TMB–DAB–Co material, we examined calbindin immunoreactivity using DAB enhanced with imidazole (Preuss et al., 1999) in six humans, three chimpanzees, one orangutan, three macaques, four squirrel monkeys and three spider monkeys. These sections were incubated in a 1:1000 dilution of the calbindin monoclonal antibody. Results obtained with DAB under these conditions were similar to those obtained with TMB–DAB–Co, and included the same pattern of species differences in staining intensity.
We also examined staining for NPNF in three humans and MAP 2 in two humans to confirm that TMB–DAB–Co immunohistochemistry yielded results consistent with our published results using DAB histochemistry (Preuss et al., 1999). NPNF immunoreactivity was examined using monoclonal antibody SMI-32 (Sternberger Monoclonals, Luthersville, MD, USA) at dilutions of 1:10 000, with sections incubated overnight at room temperature. MAP 2 immunoreactivity was examined using clone HM-2 (Sigma) at dilutions of 1:40 000, with sections incubated overnight at room temperature. Labeling was visualized using the TMB–DAB–Co procedures described above.
Double Immunocytochemistry
In single-staining procedures, the initial TMB reaction product was a deep blue or blue–green color. This changed to a dark, reddish-brown color after being stabilized with DAB and cobalt, and the reaction product became even lighter and redder during dehydration and coverslipping. We took advantage of the differences between the TMB reaction product and the TMB–DAB–Co reaction product in double-staining experiments: sections were incubated in a first primary antibody, which was visualized with TMB stabilized with DAB and cobalt, and then incubated in a second primary antibody, which was visualized with TMB alone.
In one set of experiments, we used the NPNF antibody (SMI-32) as the first primary antibody and Cat-301 as the second primary antibody, staining a total of five humans and three macaques. Sections were incubated in NPNF (diluted at 1:10 000–1:12 000) overnight at room temperature, reacted with TMB–DAB–Co, and then incubated in Cat-301 (at dilutions of 1:10–1:20 for humans and 1:10–1:40 for macaques) and reacted for TMB only. Sections were incubated in Cat-301 for 48 h at room temperature in one series of experiments, and for 7 days at 4°C in another series; human and macaque cases were included in both series so there was no confounding of species and incubation condition. We carried out chondroitin digestion to unmask the Cat-301 antigen (as described above) at the beginning of the experiment in most cases, prior to staining for NPNF, although in some cases sections were digested immediately after the NPNF step. Similar results were obtained with both procedures.
In a second set of experiments, we stained sections for NPNF and calbindin, or for Cat-301 and calbindin, using the same polyclonal calbindin antibody employed in single-staining studies. Sections from three humans and two macaques were stained for NPNF followed by calbindin. Three humans and three macaques were stained for Cat-301 followed by calbindin, and three humans and two macaques were stained for calbindin followed by Cat-301. Similar results were obtained with either order of staining. NPNF antibody was diluted by 1:12 000 for both humans and macaques, and sections were incubated overnight at room temperature. Cat-301 dilutions ranged from 1:10 to 1:20 in humans and 1:20 to 1:40 in macaques, with sections incubated for 2 days at room temperature. Calbindin antibody was diluted by 1:3600 or 1:4000 for humans and by 1:1400 for macaques, with sections incubated overnight at room temperature. Higher concentrations of calbindin antibody were used in macaques than in humans because in single-staining experiments, macaques were found to stain more weakly for calbindin than humans at a given concentration, as described in the Results.
For each case in the double-labeling experiments we prepared sections under three control conditions. Sections in the positive/negative condition were stained for the first antibody and then carried through all the steps of the second reaction, except that the second primary antibody was omitted from its incubation solution. Sections in the negative/ positive condition were exposed to the second primary antibody only. Sections in the negative/negative condition were exposed to neither of the primary antibodies. Specific staining in the positive/negative sections was observed only with the reddish-brown TMB–DAB–Co reaction product; there was no additional, specific staining with the blue TMB reaction product. This was true even when the identical species-specific secondary antibody was used in the first and second reactions, as in the case of experiments with NPNF and Cat-301, which are both mouse monoclonals. This is consistent with the report of Levey et al., using a similar double-staining protocol (Levey et al., 1986). Likewise, negative/ positive sections evinced only specific staining with the blue TMB reaction product. Negative/negative sections showed no reddish-brown or blue staining of any kind other than minor and readily identifiable peroxidase artifacts.
Microscopy, Photography and Image Processing
Sections were examined under brightfield illumination with an Olympus BX-50 microscope. Digital images were acquired with a Microlumina scanning camera (Leaf Systems, Bedford, MA, USA) and a SPOT digital camera (Diagnostic Instruments, Sterling Heights, MI, USA). Photoshop software (Versions 5 and 6, Adobe Systems, Mountainview, CA, USA) was used to subtract background images and to adjust brightness, contrast and sharpness. We also used Photoshop to represent separately the red–brown and blue elements present in double-immunostained sections. The resulting figures were particularly useful for showing the laminar and compartmental distributions of different antibodies in the same sections at relatively low magnification. In a previous publication (Preuss et al., 1999), we noted that Nissl staining and DAB labeling in Nissl-counterstained sections can be examined separately by using Photoshop's tools for splitting color channels. This simple technique proved inadequate for separating elements in our double-immunostained material, however, because the red–brown of the TMB–DAB–Co reaction product and the blue of the TMB product were relatively unsaturated, consisting of mixtures of red, green and blue hues (which varied somewhat from experiment to experiment), rather than pure hues.
In the present study, therefore, we used Photoshop's Eyedropper tool to extract unique subsets of pixels representing the TMB–DAB–Co and TMB reaction products, respectively. To take an example, we began by selecting with the Eyedropper tool a pixel from a cell or neurite that was clearly stained with TMB–DAB–Co only (i.e. a red–brown pixel). We then used the Color Range function to select pixels with RGB values similar to those of the seed pixel, adjusting the size of the envelope of selected values with the Fuzziness slider, and then copied and pasted the resulting pixel set into a new image space. We repeated this process several times, choosing seed pixels that spanned the range of RGB values representing the TMB–DAB–Co labeling. After building up an image of the red–brown labeling, we selected blue pixel sets in the same manner, building up by iteration an image of the neural elements labeled with the blue TMB reaction product. At each step in building the blue image, we chose only pixels that were not present in the extracted set of red–brown pixels. Similar results were obtained by reversing the order of selection, extracting a blue pixel subset followed by a red–brown subset. Results were improved by doing many iterations and choosing pixels covering a narrow range of RGB values at each iteration, rather than by doing few iterations that each covered broad ranges of RGB values.
Laminar Analysis
We examined the laminar distribution of Cat-301 and calbindin labeling in single-immunostained sections counterstained for Nissl substance with thionin. Selected sections were photographed and Photoshop was employed to extract the red pixels representing the TMB–DAB–Co reaction product from the blue pixels representing thionin-stained elements, using either the Eyedropper tool (as described above) or by splitting color channels (Preuss et al., 1999).
Several different schemes exist for numbering the layers of area V1 in anthropoid primates (Brodmann, 1909; Hässler and Wagner, 1965; Braak, 1976; Casagrande and Kaas, 1994; Kuljis, 1994). Most modern studies, however, follow a system based on Brodmann (Brodmann, 1909), and elaborated for macaques by Lund (Lund, 1973) (see especially Fig. 1 of that paper), and we follow that system here.
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4.5_mm anterior to the occipital pole. (B) A pair of sections stained for Nissl (left) and Cat-301 (right) taken from a more anterior level (How layer 4 is subdivided in the Brodmann–Lund system is especially pertinent to this study. Brodmann distinguished three subdivisions of this layer. The deepest stratum, 4C, is recognizable by its extremely dense cell packing. Packing density is somewhat less in the upper part of 4C than in the deeper part, so that separate upper (4C
) and lower (4Cß) sub-divisions of this stratum can be recognized (Polyak, 1957). Layer 4B is relatively cell sparse compared to 4A and 4C, and contains scattered, rather large cells (the outer solitary cells of Meynert) in addition to granule cells. Layer 4A is more densely packed with small cells than 4B and lacks the prominent outer Meynert cells. The border between 4B and 4A can usually be identified with confidence. The upper border of layer 4A, however, is often not very distinct, as the deep part of layer 3 may appear only slightly less cell dense than layer 4A (Peters, 1994). Layer 4A has been considered especially indistinct in humans (Wong-Riley et al., 1993), and perhaps even absent (Horton and Hedley-Whyte, 1984). Nevertheless, 4A was recognized in humans by Brodmann (Brodmann, 1909), and modern workers have largely followed suit (Braak, 1976; Hendry and Carder, 1993; Wong-Riley et al., 1993; Jones et al., 1994; Yoshioka and Hendry, 1995). In our thionin-stained material, layer 4A was perhaps less distinct in humans than in macaques, but could usually be delineated.
While published descriptions of macaque and human cytoarchitectonic organization provide a useful foundation for comparative studies of anthropoid primate area V1, there is at least one noteworthy variation. Le Gros Clark observed that in some New World monkey species, the solitary cells of layer 4B are not really so solitary, but aggregate to form a well-defined band (Le Gros Clark, 1942). Our observations of the New World monkey Ateles were consistent with this, although layer 4B in the New World monkey Saimiri was usually cell sparse. Some individual chimpanzees (an Old World anthropoid species) also exhibited aggregations of large cells in layer 4B.
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Cat-301 Immunoreactivity in Humans
In humans, immunocytochemistry with Cat-301 produced dense staining in the primary visual area and in neighboring cortical areas (Fig. 1
). Area V1 was distinguished in Cat-301-stained sections by its well-stratified appearance, with a prominent band of very dark staining in the middle cortical layers. The superficial border of this band had an irregular appearance, with alternating territories of light and dark immunoreactivity. These territories were present at all levels of area V1 we examined, which spanned approximately the posterior half of the area, corresponding to about the central 10° of the visual field (Horton and Hoyt, 1991). We did not observe any systematic variation in the pattern of layer 4A staining over this extent of V1, although the irregular nature of the pattern could make subtle variations difficult to detect without quantitative analysis.
Examination of Cat-301-stained sections counterstained for Nissl (Fig. 2) confirmed that the irregularly shaped territories of dark and light Cat-301 immunoreactivity were located in layer 4A. The dark-staining bands of tissue consisted of very fine neuropil, along with some larger, individually distinguishable neurites, and cell bodies with nonpyramidal morphologies (Fig. 3). Most Cat-301-immunoreactive (Cat-301+) cell bodies were small, ~10–20 µm in diameter, although scattered larger neurons, on the order of 20–25 µm, were also observed. The neuropil bands commonly extended through the thickness of layer 4A, giving off lateral branches that encapsulated territories of light staining. In some instances, vertical stacks of two or more small capsules were observed to span layer 4A. The lightly stained territories were extremely variable in size and shape. The most well-defined, capsule-like territories were typically on the order of 80–100 µm in cross-sectional diameter, although some territories were much larger.
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The dark-staining territories of layer 4A were continuous with the tissue of layer 4B, which showed very strong cell-body and neuropil labeling with Cat-301 (Fig. 3
). Layer 4B contained cells similar in morphology and size to those in layer 4A, although larger neurons (20–25 µm) were more numerous. Layer 4C also contained numerous labeled nonpyramidal cells, although neuropil labeling was lighter than in layer 4B. Layer 4Cß contained some small, lightly stained neurons and little neuropil staining. In the superficial strata, Cat-301+ cell bodies were particularly numerous in the deep part of layer 3. In addition, there was a group of large cells present in upper layer 3 and layer 2 that stained relatively completely with Cat-301, revealing elaborate, multipolar dendritic arbors (see especially Fig. 5A). In some sections, patchy or columnar aggregations of stained cells and neuropil were observed in layer 3 (Figs 1 and 2C), consistent with reports that Cat-301 stains blobs (puffs) in area V1 of primates (Hockfield and McKay, 1983; Hendry et al., 1984, 1988). There was an additional band of Cat-301+ cells and neuropil located deep in the cortex, corresponding to layers 5 and 6. Within this band was a line of very large, well-stained cells in the deepest part of layer 5 (Figs 2A–C and 4A), corresponding to the giant Meynert–Cajal cells (Braak, 1976).
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The network of dark, Cat-301+ tissue bands in layer 4A was very similar in form to the pattern of staining produced with NPNF or MAP 2 antibodies, as illustrated in Figure 4
. The distribution of staining for NPNF and MAP 2 we obtained using the TMB–DAB–Co technique in the present study closely matched the results of our previous study using an enhanced DAB technique (Preuss et al., 1999). Cat-301 Immunoreactivity in Humans Compared with Nonhuman Primates
In nonhuman primates, as in humans, immunostaining with Cat-301 yielded separate main bands of dark label in the middle and deep strata of the cortex, corresponding to layer 4B (in some cases extending into layer 4C, also), and in layers 5 and 6 (Fig. 5). However, none of the nonhuman primate species we examined showed the mesh-like pattern of darkly stained tissue bands surrounding lightly stained territories that was present in layer 4A of humans. Rather, in all nonhuman species, layer 4A appeared as a relatively light stratum, with a modest number of stained cells and dendrites.
Additional phyletic differences were noted, including strong variations in the apparent strength of Cat-301 immunoreactivity. Of the taxa examined, macaques and spider monkeys required the shortest incubation periods (1 day) and the lowest antibody concentrations (1:20–1:40) to yield strong immunostaining. In humans, strong staining required somewhat higher antibody concentrations (1:10–1:20) than those optimal for macaques; longer incubation times also resulted in enhanced staining, although strong staining was obtained in many cases with 1 day incubation periods. Squirrel monkeys showed the weakest labeling of the taxa examined, requiring long incubations (7 days) in high concentrations of primary antibody (1:4–1:8) to yield consistent staining, and even under those conditions we observed less specific staining of V1 than in humans, macaques, or spider monkeys. The great apes (chimpanzees, orangutan) also showed generally weak Cat-301 immunoreactivity, even with long incubation periods and high antibody concentrations, although individual neurons or classes of neurons were very well stained in some cases that otherwise exhibited weak staining. Strong species differences in Cat-301 staining intensity have been noted in other investigations (Hendry et al., 1988; Jain et al., 1994; Preuss et al., 1998). In addition to these general species differences in immunoreactivity, there were several marked differences in the specific laminar distribution of Cat-301 staining, particularly in the deepest layers of cortex. In macaques, and in the one orangutan examined, layer 6 was the most densely stained stratum of the cortex, and labeled cells were especially numerous in the deepest part of layer 6. In humans and chimpanzees, by contrast, layer 4B was the most densely stained layer.
Relationship of Cat-301 and NPNF Staining in Humans
As noted above, Cat-301 immunostaining yielded intermingled territories of dark and light staining in human layer 4A that resembled the pattern of labeling observed with NPNF and MAP 2 immunostaining. Double-staining studies indicated that the dark and light territories of Cat-301 immunoreactivity were coincident with dark and light zones of NPNF immunoreactivity. This is readily apparent in low-magnification photomicrographs of double-stained sections with color-processing separation of NPNF+ and Cat-301+ elements (Fig. 6A–C). At higher magnification, we observed bands of tissue containing numerous, intermingled neurites and somas that were positive for Cat-301 or NPNF elements, separated by territories that contained very few stained elements (Fig. 6D,E). Within the darkly stained territories, many Cat-301+ neurites appeared to be closely apposed to NPNF+ neurites, although we observed no un-ambiguously double-labeled neuronal elements in layer 4A. Double-stained cell bodies were observed in other layers, however, including some of the giant Meynert–Cajal cells in deepest layer 5 (not illustrated). The appearance of individual cell bodies and neurites stained for Cat-301 was consistent with that observed in single-stained preparations (Fig. 6C,D): Cat-301 stained primarily small-to medium-sized multipolar, non-pyramidal neurons and fine, diffuse neuropil in 4A, while NPNF-immunoreactive cell bodies appeared to be somewhat larger on average and included both nonpyramidal and small pyramidal profiles. The neurite complement that stained for NPNF included dendrites extending into layer 4A from deep layer 3 and from 4B; the latter included thick apical dendrites arising from large pyramidal cells, as well as the finer processes of smaller cells.
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Calbindin Immunoreactivity in Humans Compared with Other Primates
As a first step in elucidating the relationship of calbindin distribution in human layer 4A to that of Cat-301 and NPNF, we examined an extensive series of sections from humans and other primates stained with a monoclonal and a polyclonal antibody for calbindin. In all taxa examined, calbindin antibodies stained large numbers of small neurons in layers 2–4, and smaller numbers of cells in layers 5 and 6 (Fig. 7). Extensive staining of fine neuropil was also observed, especially in the upper layers, and in some taxa, dense neuropil staining outlined more lightly stained territories that presumably correspond to blobs, as reported previously (Blümcke and Celio, 1992; Hendry and Carder, 1993). Among the primates examined, however, only humans displayed irregularly shaped territories of dense immunoreactivity in layer 4A that stood out against the more moderately stained layers 3 and 4B (Figs 7A,B and 8A). These territories consisted of very darkly stained, small (~10 µm in diameter), round somas surrounded by very fine neuropil. The lightly stained territories surrounding the dark zones also contained calbindin+ cell bodies and neuropil, although these elements were less numerous and more lightly stained than in the dark-staining zones. In apes, calbindin staining in layer 4A was more uniformly distributed than in humans, and of more moderate density. Staining of chimpanzee layer 4A was comparable in density to layer 4B (Figs 7C and 8B). The single orangutan we examined was similar to the chimpanzees, although layer 4A appeared slightly less densely stained than 3 and 4B in most sections (Fig. 7D). The Old World and New World monkeys examined all exhibited a prominent, relatively uniform, label-sparse band in layer 4A, sandwiched between well-stained layers 3 and 4B (Figs 7E–H and 8C). In all species examined, the calbindin+ neurons in layer 4A had small, round cell bodies, which resemble calbindin-containing GABAergic interneurons described in other layers of area V1 (Van Brederode et al., 1990; Jones et al., 1994).
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As with Cat-301, we noted substantial differences between species in the apparent strength of staining for calbindin. Pan and Saimiri yielded the most robust staining, and Macaca the weakest, such that relatively high concentrations of the polyclonal primary antibody (1:1000–1:2000) were required to produce levels of cell staining in Macaca comparable to those obtained with lower concentrations (1:6000–1:8000) in Pan and Saimiri, and even at these higher concentrations, neuropil staining in Macaca was relatively weak. A similar relationship between species and staining intensity was observed with the monoclonal antibody.
Relationship of Calbindin Immunoreactivity to NPNF and Cat-301 Immunoreactivity in Humans and Macaques
Double-staining experiments revealed a complementary relationship between calbindin immunoreactivity and staining for NPNF and Cat-301 in human layer 4A. In all human cases examined, tissue zones that stained densely for calbindin corresponded to territories that stained lightly for NPNF or for Cat-301 (Figs 9 and 10A–C). As in our single-stained material, the dense-staining calbindin+ zones were filled with small, intensely immunoreactive neurons and neuropil. The tissue bands that stained strongly for NPNF and Cat-301 also contained some calbindin+ neurons. We observed no unambiguously double-stained elements in layer 4A, although individual cell bodies that stained for both calbindin and Cat-301 were observed in other layers, and were especially prominent in the upper part of layer 3.
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Comparison of human and macaque sections double-stained for Cat-301 and calbindin (Fig. 10D,E
) highlighted the differences between these taxa noted in single-stained sections. In humans, layer 4A was filled with intermingled territories of Cat-301 and calbindin immunoreactivity. In macaques, by contrast, layer 4A was a label-sparse band, the dominant elements being dendrites extending through this layer from somas residing in neighboring layers. Similar results were obtained comparing human and macaque sections double-stained for NPNF and calbindin (not illustrated). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Evidence for Two Alternating, Neurochemically Distinct Tissue Compartments in Human Layer 4A
Immunostaining for Cat-301 in human area V1 revealed a mesh-like pattern of staining, with dark-staining tissue bands surrounding territories of light staining, very similar to the pattern observed after staining for NPNF or MAP 2. The immunoreactive bands consisted of fine neuropil, along with some larger, more distinct dendritic branches and small-to-medium-sized nonpyramidal neurons. In double-staining experiments, Cat-301 and NPNF labeled the same tissue compartments within layer 4A. Of the primate taxa we examined, which included apes, Old World monkeys, and New World monkeys, only humans exhibited substantial Cat-301 immunoreactivity in layer 4A, and only humans showed a mesh-like distribution of staining in layer 4A.
Immunostaining for calbindin also revealed an irregular pattern of darkly stained territories in layer 4A of humans, but not in great apes, Old World monkeys, or New World monkeys. The dark zones consisted of numerous small, round cells and neuropil; lighter calbindin staining was observed outside the dark zones. Double-staining experiments indicated that the zones of dense calbindin immunoreactivity were located in the territories within layer 4A that stained lightly for NPNF and Cat-301.
Our results thus indicate that human layer 4A is composed of two alternating types of tissue compartments, one compartment being a meshwork of tissue bands that strongly express the Cat-301 antigen and NPNF, and the other consisting of calbindin-dense territories set in the interstitial spaces of the meshwork. The organization of layer 4A in human area V1 is distinctly different from that of the other primate species we examined, including chimpanzees (Pan), the animals most closely related to humans, and macaque monkeys, the primates that have been most intensively studied, as models of the human visual system. Compared to other primates, human layer 4A shows increased staining for molecules related to the M pathway.
Comparison with Previous Studies of Cat-301 Immunoreactivity in Primate V1
Most studies of Cat-301 immunoreactivity in area V1 of primates have focused on macaque monkeys (Hockfield et al., 1983; Hendry et al., 1984, 1988; DeYoe et al., 1990; Hockfield and Sur, 1990; Hockfield et al., 1990; Jain et al., 1994), although there are also reports on humans (Hockfield et al., 1990) and prosimian primates (Hendry et al., 1988; Jain et al., 1994). There are no previous published accounts of Cat-301 immunoreactivity in the visual cortex of apes or New World monkeys.
Published studies of Cat-301 immunoreactivity in macaques indicate these animals display two distinct strata of strong cell and neuropil staining, corresponding to layer 4B (or 4B plus 4C) and layer 6, the latter being most strongly stained. Staining of individual neurons or small clusters of neurons is observed in other layers. The majority of stained cells have nonpyramidal morphologies. Hockfield et al. reported that humans display a similar distribution of staining, with a very prominent upper band of staining corresponding to 4B/4C, and a deep band corresponding to layer 6, although staining of the deep stratum is weaker in humans than macaques, so that 4B/4C is the most prominent stratum in humans (Hockfield et al., 1990). They did not note substantial Cat-301 immunoreactivity in layer 4A of humans, nor any difference between humans and macaques in the appearance of 4A. Our observations of Cat-301 staining in macaques are consistent with published accounts. In humans, furthermore, consistent with Hockfield et al., we observed that staining of layer 6 was weak compared to staining of layers 4B/4C. Unlike Hockfield et al., however, we also observed territories of strong Cat-301 immunoreactivity in layer 4A of humans, which distinguished humans from the other primates we examined.
We suggest that the disparity between the present results and previously published studies with Cat-301 can be attributed primarily to the greater sensitivity of the TMB–DAB–Co technique compared to the DAB methods used in previous studies. The difference is exemplified by the greater number of stained cells visible at low magnification and the more extensive staining of dendritic trees achieved in the present study compared with other published studies using Cat-301. TMB replaced DAB as the preferred chromagen in studies of connectivity with horseradish peroxidase about two decades ago, owing to the much greater sensitivity of TMB (Mesulam and Rosene, 1979), but successful adaptation of TMB for use with immunocytochemistry was not achieved until more recently, when techniques were developed to stabilize the reaction product at the relatively high pH required for immunocytochemistry (Liang and Wan, 1989; Norgren and Lehman, 1989; Weinberg and van Eyck, 1991; Llewellyn-Smith et al., 1993). TMB also has the merit of being readily adapted for use in sequential double-labeling experiments, such as those presented here. There are, to be sure, disadvantages to TMB compared to DAB. We have found it more difficult to control nonspecific background staining with TMB than with DAB, and it sometimes produces granular artifacts. These problems are most acute when TMB is used without DAB–Co stabilization, as for instance in the second stage of double staining, because the stabilization procedure tends to clear the background. Nevertheless, for many purposes, the benefits of TMB immunocytochemistry outweigh its short-comings.
Comparison with Previous Studies of Calbindin Immunoreactivity in Primate V1
In all the primate taxa we examined, we observed numerous calbindin+ neurons in layers 2, 3, 4B, 4C and 4Cß, with scattered neurons present in layers 5 and 6. Neuropil labeling was also prominent in the upper layers of most taxa, although not in macaques. Previous studies of calbindin distribution in area V1 of primates all demonstrated cell-body labeling in layers 2 and 3, but the strength of neuropil staining and cell-body staining in layers 4–6 has varied. The laminar patterns of staining we observed are very similar to patterns observed in studies which demonstrated staining of the deeper cortical layers, in addition to layers 2 and 3 [Homo: (Hendry and Carder, 1993; Yoshioka and Hendry, 1995; Yan et al., 1997; Letinic and Kostovic, 1998); Macaca: (Van Brederode et al., 1990; Blümcke et al., 1994; Yoshioka and Hendry, 1995; Peters and Sethares, 1997); Saimiri: (Blümcke and Celio, 1992; Hendry and Carder, 1993); Callithrix: (Goodchild and Martin, 1998)].
It is noteworthy that in all the Old World and New World monkeys that we examined layer 4A stood out as a relatively light band between more darkly stained layers 3 and 4B. This is a common feature of published reports for Old World and New World monkeys. There is one discrepant report, for the New World monkey Callithrix by Goodchild and Martin (Goodchild and Martin, 1998), who demonstrated a laminar pattern of labeling very similar to other New World and Old World monkeys, but who designated the cell-sparse band as layer 4B rather than 4A.
Hendry and Carder observed that calbindin staining of layer 4A in humans differs markedly from that of Old World and New World monkeys: layer 4A is label sparse in monkeys, while humans possess irregularly shaped zones of dense calbindin staining (Hendry and Carder, 1993). Results of the present study confirm these observations, as have other reports for humans (Yoshioka and Hendry, 1995; Yan et al., 1997; Letinic and Kostovic, 1998). The clear difference between humans, on the one hand, and Old World and New World monkeys, on the other, raises the question of whether apes are human-like or monkey-like. Our observations indicate that apes differ from both humans and monkeys: apes display more staining of layer 4A than do monkeys, and thus lack a prominent label-sparse band, but the staining is weaker than in humans (being comparable only to the level of staining in layer 4B) and is not segregated into label-rich and label-poor territories. This suggests that the compartmental pattern observed in humans is a true human specialization, which evolved from a condition similar to that found in living chimpanzees.
Although we confirmed the previous results of Hendry and Carder (Hendry and Carder, 1993) regarding the distinctive appearance of calbindin staining in human layer 4A, the results of our double-staining experiments suggest a different interpretation of human layer 4A organization than that proposed by Yoshioka and Hendry (Yoshioka and Hendry 1995). They stained adjacent tangential (flattened) sections separately for NPNF and for calbindin, and concluded that NPNF and calbindin occupy complementary compartments, which is consistent with our observations of coronal sections double stained for these proteins. They maintained, however, that human layer 4A has an essentially macaque-like, honeycomb organization, whereas our results suggest there are substantial differences between humans and macaques in the spatial arrangement of neurons in layer 4A, as well as in their neurochemical phenotypes. Yoshioka and Hendry also indicated that the calcium-binding protein parvalbumin is distributed in a non-homogeneous manner in layer 4A, and localizes within the same compartments as calbindin. We have not examined parvalbumin immunoreactivity in flattened sections, but parvalbumin has a relatively uniform appearance in coronal sections through layer 4A of humans (Blümcke et al., 1990; Jones et al., 1994; Yoshioka and Hendry, 1995; Letinic and Kostovic, 1998), quite different from the irregular distribution of calbindin which can be readily seen in coronal sections.
Organization and Evolution of Layer 4A in Old World and New World Monkeys
The present results highlight the existence of substantial differences in the cellular architecture of layer 4A in humans and nonhuman primates, as summarized in Figure 11. The organization of macaque layer 4A has been very well characterized. It receives a direct geniculate projection, arising from the parvocellular layers (Lund, 1973; Hendrickson et al., 1978; Blasdel and Lund, 1983; Horton, 1984). Macaque 4A also receives afferents from layer 4Cß (Fitzpatrick et al., 1985; Yoshioka et al., 1994), itself a target of P-geniculate projections. Examination of sections cut tangential to the pial surface indicates that the sheet of geniculate-recipient tissue in 4A is punctuated by regular gaps, resulting in a honeycomb-like appearance (Hendrickson et al., 1978). The honeycomb can also be seen in tangential sections stained for CO (Horton, 1984; Fitzpatrick et al., 1985; Hevner and Wong-Riley, 1990), which presumably reflects the high level of activity of geniculostriate afferents. The pyramidal cell bodies and apical dendrites that extend upward from layer 4B into 4A, filling the gaps in the honeycomb walls, can also be visualized histochemically, using antibodies that preferentially label pyramidal cells, such as the MAP 2 (Peters and Sethares, 1991; Hendry and Bhandari, 1992) and NPNF antibodies (Hof and Morrison, 1995; Yoshioka and Hendry, 1995; Chaudhuri et al., 1996; Hof et al., 1996; Preuss et al., 1999). The affinity of these pyramidal-cell ‘cones’ (Peters and Sethares, 1991) or ‘cores’ (Hendry and Bhandari, 1992) to the M, P and K pathways has been less clear than that of the honeycomb walls, which are clearly P related. Peters and Sethares (Peters and Sethares, 1991) suggested that since these clusters of cells and dendrites are surrounded by P-geniculate afferents, they are well placed to receive P inputs. Hendry and Bhandari (Hendry and Bhandari, 1992) indicated that the clusters should be related to the M pathway, because they are rooted in layer 4B, and 4B receives input from layer 4C, which is a major target of projections from the magnocellular geniculate layers. Recent physiological evidence, however, suggests these cells receive inputs from both the M and the P pathways (Yabuta et al., 2001).
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