Department of Neurobiology and Anatomy, University of TexasHouston Medical School, Houston, TX 77030, USA
Address correspondence to Daniel J. Felleman, Ph.D., Department Neurobiology and Anatomy, University of TexasHouston Medical School, 6431 Fannin, Houston, TX 77030, USA. Email: felleman{at}nba19.med.uth.tmc.edu.
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Abstract |
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Introduction |
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V2 is characterized by a modular pattern of CO activity that differs from that in V1 by the scale of its modules and their overall organization. V2 contains a series of dense and pale stripes of CO activity that can be separated into two types of dense stripes, thick and thin, separated by pale interstripes (Tootell et al., 1983). These three CO compartments of V2 differ somewhat in their receptive field properties. The early electro-physiological studies of V2 stripe compartments emphasized the differences in receptive field properties across stripes, but it was observed that they did not support a strict segregation of color and orientation processing in V2 (DeYoe and Van Essen, 1985
). Subsequent microelectrode recording studies have emphasized the similarities in receptive field properties across V2 compartments (Levitt et al., 1994
; Gegenfurtner et al., 1996
). However, optical recording of intrinsic cortical signals demonstrated that these V2 stripes are distinguishable from each other on the basis of response selectivity, despite the functional heterogeneities within thick and thin stripes (Roe and Tso, 1995
). These results suggest that V2 stripe compartments are functionally distinct, but that heterogeneities in each stripe tend to obscure their overall differences in receptive field properties, at least at the level of individual cells.
The three CO compartments of V2 differ in their patterns of projections to extrastriate cortex. Cells in thick stripes project to the middle temporal area, MT (DeYoe and Van Essen, 1985; Shipp and Zeki, 1985
), as well as to area V3 (Felleman et al., 1997
). In contrast, cells in the V2 thin stripes and interstripes project most heavily to area V4. Recent investigations relying on random placement of retrograde tracers in V4 suggest that V4 is anatomically heterogeneous. DeYoe et al. used pairs of fluorescent retrograde tracers to demonstrate that nearby loci in V4 can receive input from topographically overlapping but segregated populations within V2 thin stripes or interstripes (DeYoe et al., 1994
). Zeki and Shipp (1989) suggested that the segregation of thin stripe and interstripe inputs to V4 is incomplete in that some regions of V4 may receive exclusive interstripe input, while other regions receive inputs from both thin and interstripes (Zeki and Shipp, 1989
). Felleman et al. demonstrated that clusters of cells within individual thin stripes could project to separate foci in V4 (Felleman et al., 1997
). Furthermore, they demonstrated that some V4 injections result in complex labeling patterns that extend across CO borders in V2. These results suggest that the pattern of V2 projections to V4 is complex and will be difficult to reveal using retrograde tracer injections alone.
We studied the organization of the V2 projections to V4 by injecting distinguishable anterograde tracers into functionally identified V2 stripes. The results indicate that V4 consists of large domains that receive V2 input largely from thin stripes or from V2 interstripes. These results also demonstrate that less dense zones within these large primary foci receive a convergence from the other complementary stripe type. Portions of these data have been presented previously in abstract form (Xiao and Felleman,1995, 1996
; Xiao et al., 1997
).
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Materials and Methods |
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Experiments were carried out in 21 hemispheres of 14 juvenile macaque monkeys (Macaca fascicularis). In preparation for surgery, the monkey was first premedicated with atropine (0.04 mg/kg, i.m.) and restrained with ketamine (1015 mg/kg, i.m.). Anesthesia was induced with Nembutal (10 mg/kg, i.v.), an endotracheal tube was inserted, and the monkey was inserted into the stereotaxic unit. Intraoperative anesthesia was provided by Isoflurane (0.52.0%) which was supplement by Nembutal as needed. A stainless steel recording chamber was implanted surrounding a craniotomy overlying the foveal and parafoveal portions of V2, V4 and part of V1. Buprenex (10 mg/kg, i.m.) provided post-surgical analgesia and prophylactic antibiotics (100 000 units procaine penicillin G) were given for 7 days, after which weekly recording sessions began.
Physiological Preparation
For recording sessions, the monkey was restrained with ketamine (20 mg/kg, i.m.), intubated with a cuffed endotracheal tube, catheterized for i.v. infusion, and supported on an air table using a custom fabricated head holder. The recording chamber was opened using aseptic technique. Recording sessions were carried out under Sufenta anesthesia administered via a continuous i.v. infusion (612 µg/kg/h) in lactated ringers. Paralysis was induced and maintained by infusion of Pavulon (pancuronium bromide; 0.05 mg/kg/h, i.v.). The monkey was respired with a Sechrist infant ventilator providing a mixture of air and oxygen. End-tidal CO2 was measured continuously (Ohmeda 5200 Medical Gas Analyzer) and maintained at 3.74.3% by adjusting the respiratory rate and volume. The EKG and transcutaneous pO2 were monitored continuously. The animal's body temperature was maintained at 37.5°C by a feedback-controlled electronic heating blanket (Harvard). The eyes were fitted with zeropower contact lenses following cycloplegia and mydriasis produced by applications of atropine (2%) and neosynephrine (2.5%). Each eye was then brought into focus on the tangent screen (114 cm distance) using trial lenses as determined by retinoscopy. The positions of the foveae were determined with a reversible ophthalmoscope.
Visual Stimulation
Visual stimuli were generated using a SGI workstation which could display flashed and moving spots, bars and gratings with various luminance and chromatic contrast. A 20 in. Trinitron monitor was used to display luminance and chromatic gratings with a mean luminance of 826 cd/m2. The luminance and chromaticity of the stimulus were calibrated using a Tektronix J17 LumaColor meter with a J1803 luminance head and a J1820 chromaticity head, and different chromatic gratings were adjusted for equal luminance (within 3%).
Optical Recording
The intrinsic optical signal, derived from 630 (± 15) nm light reflected from the exposed cortical surface, was recorded during various visual stimulation conditions according to the methods developed by Grinvald and his colleagues (Frostig et al., 1990, Tso et al., 1990
). A hydraulically sealed stainless steel chamber was implanted surrounding a large craniotomy (up to 1 cm2) and was filled with sterile silicone oil during the recording sessions. An anti-reflection-coated glass window provided optical access to the cortical surface. The brain surface was imaged using a slow-scan CCD array camera (Photometrics Ltd) that provided a wide dynamic range and low noise signal. The cortical surface was imaged using a tandem lens system that provides a numerical aperture of 0.4 NA at 12.5x, as described by Ratzlaff and Grinvald (Ratzlaff and Grinvald, 1991
). Thus it was possible to acquire enough light to achieve acceptable levels of photon noise in brief exposures (~100 ms). The image acquisition/analysis was done using IMAGE software (Biological Detection Systems, BDS; now owned by Oncor). The data acquisition and visual stimulus presentations were synchronized to respiration so that image sequences could be averaged across many trials and compared across stimulus conditions.
The data consist of a series of 810 images of the cortical surface (focused 300600 µm below the surface), beginning before stimulus presentation and continuing 23 s into the stimulation period. An interstimulus interval of 10 s allowed cortical activity to return to baseline conditions. These image sequences were repeated 50100 times per stimulus and the time-synchronized frames were averaged to reduce noise. Differential images were calculated to visualize the functional modules, as described below.
The CO blobs in V1 and the CO thin stripes in V2 were located using their preferences for low spatial frequency, isoluminant chromatic gratings. In these experiments, an average response was calculated during the binocular presentation of red/green or blue/yellow isoluminant gratings of different orientations (square wave, 0.25 cycles/deg, drifted at 1 cycle/s, with a mean luminance of 826 cd/m2, orientations included: 0°, 90°, 45°, 135°). The average response to achromatic luminance gratings of high spatial frequency and different orientations (sinusoidal, 2.0 cycles/deg, 2.0 cycles/s, 826 cd/m2 mean luminance, ~95% contrast, orientations included: 0°, 90°, 45°, 135°) was subtracted from the average color response. This difference was divided by the average background response to generate the final differential image. Thick CO stripes were identified by two methods. First, thick stripes were revealed by comparing images acquired during stimulation with low-contrast luminance gratings (0.25 cycles/deg, drifted at 2.5 cycles/s, 7% contrast) with images acquired during high-contrast, high spatial frequency stimulation. Second, recent works in squirrel monkeys (Malach et al., 1994) and macaque monkeys (Roe and Tso, 1995
) have shown that V2 thick stripes and interstripes contain a robust representation of orientation that is lacking in thin stripes and thus could be diagnostic of stripe compartments. To reveal orientation selectivity, two pairs of gratings with orthogonal orientations were compared. The pattern of ocular dominance in V1 was revealed by the differential image of stimulating left eye versus right eye and was used to determine the V1/V2 border.
Tracer Injections and Tissue Processing
Tracer injections were made into V2 using the vascular pattern and functional images from prior optical recording for guidance. The locations of the injections relative to V2 compartments were finally determined on V2 sections stained for CO. In each case, iontophoretic injections of biotinylated dextran amine (BDA, 10% in 0.01 M phosphate buffer, pH 7.4; 6 µA for 1030 min), Phaseolus vulgaris leucoagglutinin (PHA-L, 2.5% in 0.05 M Tris-buffered saline, pH 7.4; 5 µA for 20 min), and rhodamine-conjugated dextran amine (FluoroRuby or FR, 10% in 0.01 M phosphate buffer, pH 7.4; 8 µA for 1030 min) were made into V2 using glass micropipettes with internal tip diameters of 1530 µm. After injections were completed, the monkeys were kept alive for an additional 16 days to allow the tracers to be transported anterogradely to V4. Previous reports have shown that these tracers work better as anterograde markers. Only sporadically and in a pathway-dependent fashion did these tracers performed well as potent retrograde markers
At the termination of the anatomical transport period, the monkey was deeply anesthetized with Nembutal (75100 mg/kg, i.v.) and perfused intracardially with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). In most cases the fixative was washed out with phosphate buffer containing a graded series of glycerin (010%). The perfusion usually was brief (810 min) to permit cortical unfolding before sectioning. In these cases, the brain was removed from the skull and the occipital operculum was dissected by cutting along the fundus of the lunate sulcus (LS) and inferior occipital sulcus (IOS), as well as across the stem of the calcarine fissure. This tissue block was then unfolded slightly, gently pressed between glass slides, briefly post-fixed in the final cryoprotective solution, and later sectioned in the tangential plane. The prelunate gyrus, superior temporal sulcus (STS) and adjacent posterior inferotemporal cortex were removed from the remaining hemisphere by cutting along the fundus of the STS and the fundus of the occipitotemporal sulcus (OTS). These cuts were then connected across the inferotemporal cortex at the level of the anterior middle temporal sulcus (AMTS). This tissue block was then removed from the hemisphere by gently undercutting the remaining white matter. This tissue block was then progressively unfolded by gentle removal of the white matter and careful unfolding of the prelunate gyrus, STS and posterior temporal lobe (Felleman et al., 1997). This block was also gently pressed between glass slides and was post-fixed in the cryoprotective fixative solution. In two cases, the prelunate gyrus, STS and temporal lobe were not physically unfolded but were instead sectioned in the horizontal plane.
Frozen sections were cut at 31 µm (32 sections/mm) for horizontal sections and at 4050 µm for tangential sections. For the demonstration of fluorescently labeled cells, unstained sections were mounted on subbed slides, air-dried and coverslipped. The pattern of CO activity in V1 and V2 was demonstrated according to Wong-Riley and Carroll (Wong-Riley and Carroll, 1984). Briefly, free-floating sections were incubated in a large volume of oxygenated reaction mixture for 524 h at 37°C. Sections were then washed, mounted on subbed slides, and air dried several days before dehydration and coverslipping. The locations of CO dense and pale regions in V1 and V2 were determined using a computer-interfaced microscope scoring system or image analysis system.
In V2, CO thick stripes were distinguished from thin stripes using several different methods. First, the distribution of immunoreactivity to CAT-301 was used to distinguish the densely reactive thick stripes from the weakly reactive thin stripe compartments. CAT-301 was localized using an antibody provided by Dr Susan Hockfield and reacted according to the protocol published by DeYoe et al. (DeYoe et al., 1990). Briefly, free-floating sections were incubated for 1272 h in the monoclonal antibody CAT-301 (full strength supernatant). Secondary antibody (rabbit anti-mouse conjugated with HRP) was applied for 26 h at a dilution of 1:50 or 1:100. DAB was then used as the chromagen.
For tangentially cut V4, multiple sections stained for different tracers were interleaved with each other so that the differences in laminae among them were minimal. For horizontally cut V4, the sections stained for different tracers were interleaved with sections stained for SMI32, degenerating fibers and/or myelin. BDA was visualized with an avidin biotinperoxidase complex using the Vectorstain ABC Elite kit. Sections were incubated in the ABC solution (2 drops avidin DH and 2 drops biotinylated horseradish peroxidase per 5 ml 0.05 M KPBS) overnight at 4°C. Sections were then processed for visualization using DAB (0.05% in 0.1 M PB with 0.015% H2O2) as the chromagen (Veenman et al., 1992).
PHA-L and FluroRuby were visualized using the protocol of Dolleman-Van der Weel et al. (Dolleman-Van der Weel et al., 1994). Sections were incubated overnight in, respectively, a goat anti-PHA-L (1:1000 to 1:4000 in Tris-buffered saline with 0.3% Triton X100) or rabbit anti-TRITC antiserum (1:1000 to 1:4000), followed by incubation in IgG raised in donkey against goat or in swine again rabbit (1:50). The sections were then incubated with a peroxidaseanti-peroxidase complex (PAP), raised in goat (1:600) or in rabbit (1:800) for 1 h. Finally, sections were reacted with filtered DAB solution (0.05% in 0.05 M Tris with 0.015% H2O2).
All sections stained for BDA, FR or PHA-L were later intensified using a protocol suggested by J.B. Levitt (personal communication). Mounted and air-dried sections were dehydrated and placed in xylenes for 4 days. They were then rehydrated and placed in 1.42% AgNO3 at 56°C for 1 h, 0.2% HAuCl4 for 10 min and 5% Na2S2O3 for 5 min. A rinsing in dH2O for 15 min was carried out before and after each reaction. Finally sections were dehydrated and placed in xylenes for 12 days before coverslipping.
In one case, the posterior third of the corpus callosum was sectioned 7 days prior to killing the monkey to identify the border between area V4 and V3A. Degenerating fibers and terminals following transection of the corpus callosum were demonstrated using the technique of Wiitanen (Wiitanen, 1969) and myeloarchitectonic borders were visualized using the Gallyas method (Gallyas, 1969
). The pattern of neurofilament antibody SMI32 immunoreactivity was used to delimit the borders of several cortical areas according to the description of Hof and Morrison (Hof and Morrison, 1995
).
Anatomical Data Acquisition and Analysis
To achieve the highest accuracy possible in the anatomical methods, a computer-interfaced microscope (Zeiss Axioskop) was used to record the locations of labeled cells, terminal axons, blood vessels, myeloarchitectonic borders and edge lines using the Neurolucida software package (Microbrightfield, Inc.). In hemispheres cut in the horizontal plane, a series of sections for each type of label were generally scored at a 0.250.5 mm intervals. Individual series of tangential sections were scored at intervals of 150200 µm. Distributions of CO, BDA, PHA-L and other macroscopic patterns were also imaged using a 24-bit, high-resolution CCD camera on a light box or microscope or using a 30-bit flatbed scanner. Custom software running on a Unix workstation (Silicon Graphics Indigo2 R10000) allowed interactive alignment of scored sections to each other or to images of stained sections. Radially aligned blood vessels and other fine edge landmarks were used to align sections. The same software allowed for the reconstruction of the three-dimensional relationships among labeled structures and for the comparison of these markers to images of cytochrome oxidase-stained sections and to other section types.
The occipital block was processed for CO (Wong-Riley and Carroll, 1984) to identify CO blobs in V1 and the stripe compartments of V2. These sections allowed for the comparison of optical activation to the CO structure of V2. The occipital block was also processed for the demonstration of injection sites in V2, lateral connections within V2 and retrograde/anterograde transport to V1. The analysis of these connections will be the subject of another paper. The prelunatetemporal lobe block was treated similarly, to demonstrate connections with areas V4, V3A, V4A, MT and/or V4t. In two cases, the prelunatetemporal lobe block was sectioned in the horizontal plane as this allowed V2 axonal terminations to be compared to the pattern of SMI32 immunoreactivity, and also to the pattern of interhemispheric connections.
In most cases, the distribution of axonal labeling in the prelunate gyrus was scored by hand for all clusters at 200x or 400x magnification. Within the terminal field, the vast majority of axons were terminal axons displaying clear boutons (Rockland, 1992). Therefore the distribution of the scored axons approximates that of the terminals. To obtain a quantitative representation of the distribution of label in the terminal field, the area of interest was divided by a grid of 0.1 x 0.1 mm boxes. Within each box the length of all axons was calculated for each tracer. Since the labeled axons tended to form clusters, it was important to analyze the relationship between clusters. To define clusters automatically, the distribution of the axon length for each tracer was passed through a threshold. The threshold was set so that all the boxes above the threshold contained 90% of the total length of labeled axons across the whole grid. That is, 10% of the labeled axons in the boxes of low density were discarded. A cluster was composed of all the neighboring boxes above the threshold. The coordinate for the center of mass for each cluster was calculated as follows:
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Here Li represents the summed length of axons within the ith box; the Xi and Yi represent the x and y coordinate of that box respectively. The center of mass was then calculated for each cluster.
To provide an additional metric of the extent to which the distributions of two different traces were segregated, DeYoe et al.s segregation index (SI) was modified (DeYoe et al., 1994). First, clusters from one tracer (e.g. tracer A) were defined as above. Second, for each cluster, the total axon length was calculated for each tracer. To reduce the artifact introduced by unequal tracer sensitivities, this analysis used a relative length measure for each tracer in a cluster. Thus
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Fourth, the SI for all the clusters defined by tracer A was calculated as
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SItotal is equal to 0 when clusters defined by the two tracers are completely overlapping but is equal to 1 when they are completely segregated. This modified SI is less affected by the unbalanced sensitivity of the two tracers than the original SI. However, in cases with weak labeling of one or both tracers, the SI is still exaggerated inadvertently. Also, this SI captures the spatial relationship between clusters defined by different tracers but does not take into account the distribution of labeled axons within the clusters.
In three other cases the high density of labeled axons prevented the accurate scoring of axonal density. In these cases, quantitative imageprocessing techniques were applied to high-resolution, flat-fielded images of the labeled sections to estimate the density of labeled axons in these unscorable regions. Regions of high scoring density and medium scoring density were used to form a linear regression between axon length and optical density. This regression was then used to predict axon density in regions of high optical density. This method generally accounted for 6080% of the variance in the regression for optical density versus axon density.
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Results |
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Optical recording of intrinsic cortical signals was used to derive functional maps of area V2. These maps served to guide injections of multiple neuroanatomical tracers into adjacent thin stripe and interstripe compartments. In each case, one anterograde tracer was aimed at a chromatic zone, a presumptive thin stripe. Another injection was aimed at a non-chromatic zone, a presumptive interstripe, and the third one was aimed at another chromatic or non-achromatic zone. After tissue processing, the locations of the injection sites were carefully examined on histological sections stained for CO. The identification of CO dense stripes as thin or thick was based on the width of the stripes and/or the immunoreactivity to antibody CAT-301. The injections straddling the border between a thin stripe and an interstripe were excluded from the analysis. Eleven out of 14 injections aimed at a chromatic zone hit a thin stripe. However, only two of them were restricted to a thin stripe and gave rise to acceptable labeling in V4. Thus, only two pairs of injections one at a thin stripe and another at an interstripe were used to study the relationship between the projections from different V2 compartments to V4. In addition, five pairs of interstripe injections, without diffusion to any thin stripe, were used to study the projections from the same V2 compartments to V4. In V4, each injection produced a relatively large primary focus and multiple smaller secondary foci. The primary focus was defined as the cluster with the largest total length of labeled axons. Table 1 lists the size of the injection, the total area of all the projection foci in V4 and the size of the primary focus for each of the injections used in the paper.
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Figure 1 shows one case (9703R) in which the pair of injections were successfully restricted to adjacent thin and interstripes. Figure 1A
shows the differential image of part of the dorsal operculum obtained during chromatic versus luminance stimulation. The dark zone in V2 was most sensitive to the chromatic stimulus, indicating it was the chromatic module in a thin stripe (Roe and Tso, 1995
). FluoroRuby (FR) was injected into the dark zone and BDA was injected at the pale zone ~1 mm medial to the FR injection. In this figure, as in Figures 2 and 3
, green represents the injection site or axons labeled by BDA, while red signifies the FR injection site and axon terminals, and yellow represents where the two labels overlapped. Finally, white represents the PHA-L injection site or labeled axons.
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The projections to V4 from the BDA injection (Fig. 1E) and the FR injection (Fig. 1F
) consist of labeled axons and boutons. Figure 1G
shows the projections from both injections on the unfolded sections including parts of the lunate sulcus, inferior occipital sulcus, superior temporal sulcus and prelunate gyrus. There was a weak projection to the region of MT from the BDA injection only. Both injections produced modest projections to V3A and robust projections to V4. The projection pattern in V4 outlined by the dashed square is shown at higher magnification in Figure 1H
. In V4, the primary focus from the interstripe injection (BDA) was ~3.59 mm2, ~1.7 times as large as the thin stripe primary focus (2.15 mm2). These two primary projection foci were segregated from each other by a gap of ~1 mm. The primary focus from the thin stripe injection was located antero-ventral to that from the interstripe injection, with a center of mass to center of mass distance of 3.7 mm. The total area of all the foci from the interstripe injection was 5.06 mm2, whereas that from the thin stripe injection was 3.36 mm2. The size and number of foci and the total area occupied by each tracer depended on the sensitivity of the tracer, injection size and other factors. However, the center-to-center distance is relatively independent of these factors. The segregation index (SI) was 0.75 in this case.
In addition to the large-scale segregation of the thin stripe and interstripe primary foci, there was some convergence of a secondary focus from the interstripe injection (BDA) in the region of the thin stripe primary focus. This projection is viewed as a specific functional architecture rather than a stray projection for several reasons. First, the BDA injection was ~0.5 mm in diameter and its center was located 0.9 mm from the border of the nearby thin stripe. So the secondary focus of BDA in the primary focus of FR is not due to the diffusion of the tracer from V2 interstripe to thin stripe. Second, this secondary focus is located within a weak zone in the thin stripe primary focus. This insertion of a secondary focus into a weak zone in the primary focus from the other stripe compartment was only observed following thin stripeinterstripe pairs whereas dense overlap was observed following dual interstripe injections (see below).
Case 2
In another case (9601R), BDA was injected into a chromatic zone in V2, and FR and PHA-L were injected into two different parts of the pale zone in the differential image from chromatic versus luminance stimuli (Fig. 2A). Based on the sections stained for CO (Fig. 2B
), those for BDA or PHA-L, and unstained sections examined under fluorescent light, the BDA injection (0.25 mm in diameter) was found to be restricted to a thin stripe. The PHA-L and FR injection sites were located at two sides of a thick stripe. In this case, the identification of thin and thick stripes was based on the width of the CO dense stripe (Fig. 2B
) and Cat-301 staining (not shown). Figure 2C
shows the patterns of BDA and FR labeling in the unfolded occipito-temporal sections. The dense labeling of FR in the STS likely reflects the projection from thick stripes to MT. The sparse labeling of both tracers close to the crown of the STS may reflect the weak projections from both thin and interstripes to area V4A. The dense labeling inside the dashed square reflects the projections to V4 and V3A and is shown in detail in Figure 2D
. As in the previously described case, the more medially situated projection field is believed to correspond to area V3A and is excluded from further analysis here.
In V4, the primary focus from the thin stripe injection (BDA) was completely segregated from the primary focus from the interstripe (FR) injection by a gap of 1.3 mm (Fig. 2D). The primary focus from the thin stripe injection was relatively small (3.23 mm2) and located antero-dorsally from the interstripe primary focus (7.29 mm2). Their center-to-center distance was 4.5 mm. The spatial relationship between projections from thin and interstripe injections was strikingly similar to that observed in the previous case, even though the assignment of tracers to stripe compartments was opposite between these two cases. In both cases, the injections at thin stripes gave much weaker projections in V4 than those at interstripes, regardless of which tracer was used. In addition to this global segregation of thin stripe and interstripe projections to V4, there was also a small degree of convergence between these projections. Like the previous case, a secondary focus from the interstripe injection (FR) appeared in the weak zone in the thin stripe primary field, and no diffusion of FR injection into the nearby thin stripe was found. In addition, in this case, an insertion of a thin stripe secondary focus (BDA) into a weak zone in the interstripe primary focus was also observed. This insertion is also unlikely to have been caused by the diffusion of BDA injection into adjacent interstripes for the following reasons. Firstly, on each V2 section with clear CO stripes, the BDA injection was located at the center of a thin stripe. Given the tiny size of this injection, the diffusion, if any, must be too small to account for the inserting secondary focus. Secondly, the insertion occurred at the weak zone within the interstripe primary focus, in contrast to the peak-to-peak overlap between primary foci from a pair of interstripe injections (e.g. FR versus PHA-L in this case, see below).
The segregation index (SI) between BDA and FR was 0.58 in this case and was smaller than that seen in the previous case. The smaller SI probably resulted from more overlap between the interstripe primary focus and a thin stripe secondary focus. However, the SI is still larger than those seen in cases of dual interstripe injections.
The segregation in terminal fields in V4 described above could reflect the segregation of distinct functional streams in V4, or might depend primarily on retinotopic differences between the injections. To distinguish these possibilities, we compared the projections from two injections into the same or sequential interstripes. Figure 2E shows the labeling of PHA-L (white) and FR in V3A and V4, from the same area indicated in Figure 2D
. Both tracers were injected at the border between a thick and an interstripe as shown in Figure 2B
. PHA-L labeling was much more sparse than that of FR because PHA-L was less sensitive than FR. In V4, however, the primary focus of PHA-L labeling overlapped with that of FR, with a center-to-center distance of 0.6 mm. The segregation index between PHA-L and FR was 0.29. This was in clear contrast to the segregation seen between BDA and FR (Fig. 2D
), indicating that the segregation in Figure 2D
reflected a difference in functional architecture rather than retinotopic differences between the two injection sites.
Case 3
In this case (9704R), FR and BDA were injected into sequential interstripes, with a thin stripe between them. By comparing sections showing the injection sites with adjacent sections stained for CO, it was confirmed that both injection sites were <1 mm in diameter and were restricted to the interstripes flanking a thin stripe. However, to illustrate the clear pattern of CO stripes, Figure 3A shows a CO section that is 1 mm deeper than the injection sites. The locations and extents of the injection sites were superimposed on this deep section by carefully aligning all the operculum sections based on blood vessels. Their projections in V4 are shown in Figure 3B
. As in previous figures, green represents the BDA labeling, red represents the FR labeling, and yellow represents the overlap between the two labels. The BDA primary focus was densely labeled, and the axon density of BDA in dense portions of this field had to be estimated from the calibrated optical density. Although the FR labeling was weak, it overlapped with the peak of BDA labeling in V4. The center-to-center distance between the BDA and FR primary foci was 0.4 mm. The segregation index between BDA and FR was 0.22, thus indicating the high degree of overlap of these two projection patterns.
Analysis Across All Cases
In total, it was confirmed by CO staining that two pairs of injections were made into different V2 stripes and five paired injections were made into interstripes. Among the five pairs of interstripe injections, three pairs flanked a thin stripe as in Figure 3A, one pair flanked a thick stripe shown in Figure 2A
(PHA-L versus BDA), and one pair hit the same interstripe (not shown). Figure 4A
shows the average center-to-center distance between the pair of primary V4 foci, either from different stripes (thin versis inter; n = 2) or from interstripes (inter versus inter; n = 5). The center-to-center distance between the primary V4 foci from the different V2 stripes were significantly larger than that from like-stripes (P < 0.0001, t-test). Figure 4B
shows the average segregation index (SI) for these two groups of injection pairs (see Materials and Methods). Again, the SI for projections from different stripes (thin versus inter) was significantly larger than that from like stripes (inter versus inter; P < 0.05, t-test). Both analyses therefore confirmed that adjacent thin stripe and inter-stripe compartments of V2 make largely divergent projections in V4, whereas the adjacent interstripes make largely convergent projections.
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We assigned the clusters medial to the dashed square in Figure 1G and that medial to the dashed line in Figure 2D
as V3A for three reasons. Firstly, in 8 out of 11 cases cut tangentially, these clusters were at relatively constant locations and separable from clusters in V4, even though the locations of V4 clusters varied due to the retinotopic difference of injection sites (compare Figs 1G and 2C
). This consistent location seems to correspond to the location of vertical meridian mapped electrophysiologically (Van Essen and Zeki, 1978
; Gattass et al., 1988
), indicating the border between V3A and V4. Secondly, the occipito-temporal block of one of the cases with corpus callosotomy was cut horizontally (case 9704L). The callosal pattern was revealed by degeneration staining and mapped to an adjacent section stained for BDA projection from a V2 interstripe, shown in Figure 6A
. The white dots represent the locations of degenerated callosal terminals, with their size representing the relative density of these terminals. The black patches represent the clusters of BDA labeled terminals. Based on electrophysiological mapping (Van Essen and Zeki, 1978
), the border between V4 and V3A was assigned to the medial edge of the callosal patch, as marked by an arrow in Figure 6A
. The BDA-labeled cluster indicated by an arrowhead was clearly located within V3A. The location of this V3A cluster roughly corresponds to the location of the putative V3A clusters in Figures 2 and 3
. Thirdly, Figure 6B
shows scored BDA labeling on a horizontally cut section from another case (9502R), in which an interstripe was injected. Here, short segments represent individual axons, whereas the solid patches represent dense clusters of terminals. A nearby section stained for SMI32 is illustrated in Figure 6C
. According to Hof and Morrison, area V4 contains significantly more SMI32-immunoreactive neurons in the supragranular layers as compared to area V3A (Hof and Morrison, 1995
). In Figure 6C
, the arrow points to the location where there is a sharp change in SMI32-immunoreactive neurons in the supragranular layers. Thus this location probably corresponds to the border between V3A and V4. The labeled axons medial to the arrow were most likely located in area V3A. These three lines of evidence support the distinction of a projection field in V4 from a more medially situated projection field in V3A.
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Discussion |
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Figure 7 summarizes our results on the projections to V4 from a pair of injections localized to a V2 thin stripe and interstripe. In most of our cases, an injection of an anterograde tracer in V2 generated a primary focus and multiple secondary foci in V4. In the two cases with one injection in a thin stripe and the other injection in the adjacent interstripe, the primary foci from the two injections were separated by a gap of ~1 mm. In each case, the primary focus from the thin stripe injection was anterior to and half the size of the primary focus from the interstripe injection. The distance between the centers of the two primary foci was ~4 mm. In addition, there was secondary focus from a V2 compartment overlapping with the weak zone within the primary focus from the opposite V2 compartment, which we term an insertion. These features of the relationship between the projections from different stripes were observed in another case that is neither shown nor included in the quantitative analysis because of the poor CO staining on V2.
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The segregation between the projections from different V2 compartments to V4 cannot be explained by the retinotopic difference between the pair of injections. In five cases with pairs of injections outside thin stripes, the two primary foci, both from the interstripe, were largely overlapping and/or with center-to-center distances <1 mm. Both the center-to-center distance between primary foci and the segregation index between the overall projections were significantly smaller in the cases with dual interstripe injections compared to the cases with injections in a thin stripe and an interstripe. In the two cases with segregated primary foci, the injections at the thin stripe and interstripe were either adjacent to each other (case 9703R, BDA; case 9601R, PHA-L), or with an interstripe and a thick stripe between them (BDA and FR in case 9601R). In the five cases with overlapping projections from pairs of interstripes, three pairs of injections flanked a thin stripe, one pair flanked a thick stripe and one pair was in the same interstripe. Roe and Tso have reported that the visual field overlap between a pair of interstripes is smaller than that between an adjacent thin stripe and interstripe within a stripe cycle (Roe and Tso, 1995) (their Fig. 7
). From this point of view, for the two pairs of thin stripe versus interstripe injections in the current study, the retinotopic difference between the injection sites was smaller, compared to the four pairs of injections at sequential interstripes.
The retinotopic difference between a pair of injection sites also depends on the magnification factor, which decreases from fovea to parafovea. With the same physical distance, a pair of injection sites at the fovea has a smaller retinotopic difference than a pair at the parafovea. It is important to examine whether the pairs of the dual interstripe injections were closer to fovea than those of thin stripe versus interstripe injections in the current study. After mapping all the injections across different cases onto one operculum, we found that the pairs of thin stripe versus interstripe injections were scattered within the range spanned by the pairs of dual interstripe injections. On average, these two groups of injections were at locations with comparable magnification factors. Therefore, the larger segregation between the projections from the thin stripe and interstripe reflected the distinction between these two compartments, whereas the shift in the projection fields from a pair of sequential interstripes resulted from the topographic shift from one interstripe to the next interstripe.
Comparison with Retrograde Tracer Injections of V4
The current results are largely if not completely consistent with the results of retrograde tracer injections to V4 that labeled specific V2 CO compartments. DeYoe et al. and Zeki and Shipp both recognized that individual retrograde injections to V4 could label a population of interstripes in V2 (Zeki and Shipp, 1989; DeYoe et al., 1994
). These two studies differed in that Zeki and Shipp failed to make a V4 injection that only labeled V2 thin stripes while DeYoe et al. illustrated a case that contained almost exclusively thin stripe labeling after a single V4 injection. Similarly, Felleman et al. made multiple retrograde injections in V4 and demonstrated cases of multiple thin stripe or multiple interstripe labeling (Felleman et al., 1997
). These results suggest that V4 contains V2 thin stripe and interstripe-recipient modules. While these retrograde studies reached these conclusions based upon indirect evidence, the current anterograde results based on injections into physiologically identified V2 stripes provide the first direct evidence of the segregation of V2 thin stripe and interstripe projections to V4. These results thus provide evidence of the size and spacing of V2 thin stripe and interstripe projections to V4.
Convergence Between the Primary and Secondary Foci of the Projections in V4 from Different V2 Stripes
A second prominent aspect of the organization of the V2 projection to V4 is the convergence of smaller secondary foci from one compartment into weaker zones in the other compartment's primary focus. This pattern of convergence was observed for interstripe insertions into thin stripe primary foci and also for thin stripe insertions into interstripe primary foci. In each case of interstripe or thin stripe insertions, the secondary focus was ~500 µm wide while the opposite primary focus was 14 mm wide. As described in the results, these insertions were unlikely to have been caused by the diffusion of the injections into the adjacent stripes. This organization suggests that convergence of different functional streams might occur in at least two ways. Convergence might occur at the level of localized clusters of neurons that can receive direct input from both thin-stripe and interstripe streams. Because the average basal dendritic field width for V4 pyramidal cells is nearly 400 µm (Lund et al., 1993), a large number of neurons in V4 could sample from intercalated projections. Alternatively, convergence between functional streams could occur through lateral, intrinsic connections linking modules of different functional streams. The wide distribution of intrinsic connections in V4 suggests that considerable cross-talking could occur between different modules, but these patterns have not yet been linked to specific receptive field properties or to specific anatomical connections (Yoshioka et al., 1992).
Rockland (1992) has shown that a single axon entering V4 from V2 gave off 35 axon arbors that are 0.2 mm in width and 0.40.8 mm apart (Rockland, 1992). In the current study, the primary focus and the inserting secondary focus generated by an injection were separated by a gap >1 mm. Therefore, these two foci may originate from different populations of cells within the injection site, with few cells projecting to both foci. In most of our cases, an injection with a diameter of 0.151.0 mm gave rise to one or more secondary foci separated from the primary focus by a gap >0.8 mm. Thus, any spot in V2 consists of different populations of cells projecting to separate foci in V4.
Modular Organization of V4 and its Functional Implication
Our results, together with the previous studies with injections of retrograde tracers in V4 (DeYoe et al., 1994; Felleman et al., 1997
), suggest that V4 consists of domains dominated by input from thin stripes or interstripes. The thin stripe recipient domain is smaller than the interstripe recipient domain, as revealed by the sizes of the primary foci. To reveal the global organization of these domains, an ideal approach would be to fill all the thin stripes with one tracer, and fill all the interstripes with another tracer, and then study their labeling in V4. However, this approach is technically impossible because only a small portion of V2 is on the surface of the operculum and subject to the guided injection.
A model of the global organization of V2 afferent in V4, largely based on the superimposition of 11 interstripe projections and 2 thin stripe projections (Fig. 5), is illustrated in Figure 8
. According to this model, V4 consists of alternating bands dominated by V2 inputs from interstripes or thin stripes. Each band consists of multiple clusters with dense V2 projection. The weak zones (shown as gaps in Fig. 8
) receive projections from the opposite stripes of V2. The interstripe recipient band is ~35 mm wide while the thin stripe recipient band is ~12 mm wide. This difference in width was not estimated from Figure 5
, since Figure 5
consists of unequal number of projections from thin stripes (2) and interstripes (11). Instead, the difference in width was derived from two pairs of projections from different stripes shown in Figure 1 and 2
. In both cases, the primary focus from the interstripe was at least 60% larger than that from the thin stripe, even though the assignment of the tracers to stripes was reversed between these two cases. The relative larger size of the interstripe-recipients is consistent with Zeki and Shipp's observation that injections of retrograde tracers in V4 always labeled interstripes but sometimes spared thin stripes. The width of a cycle is ~46 mm, consistent with the 5 mm estimated from previous studies in which pairs of retrograde tracers were injected into V4 (DeYoe et al., 1994
). However, this width could be smaller at peripheral portions of V4 since V4 is narrower at larger eccentricity (Gattass et al., 1988
). According to our model, the bands in V4 are wider than the stripes in V2, consistent with the trend of the functional modules revealed by the optical recording (Ghose and Tso, 1995
).
|
Roe and Tso have shown that V2 contains multiple interleaved visual maps, one for each type of stripes (Roe and Tso, 1995). If this is also true for their V4 targets, our model can explain the finding by Van Essen and Zeki that V4 contained multiple representations of some parts of the visual world (Van Essen and Zeki, 1978
). While the mapping studies on anesthetized monkeys did not show a vertical meridian crossing the prelunate gyrus (PLG), one study on awake monkeys reported such a vertical meridian. The PLG part of this meridian could be the vertical meridian of the thin stripe recipient band, which was on the PLG in Figure 5
.
As revealed by most of the previous physiological studies, between the two substreams, the blobthin stripe substream contains more color-selective cells, whereas the interblob interstripe substream contains more orientation-selective cells. If the projection from V2 to V4 provides the major driving force for the activities of V4 neurons, one would expect V4 to contain separate modules predominantly selective for color or orientation according to our model. Physiological studies have demonstrated the existence of such modules in V4. Zeki found a module with 84% of wavelength selective cells and another module with only 19% of such cells (Zeki, 1983). Since only 20% of orientation-selective cells were also wavelength selective in his study, it is likely that most of the orientation-selective cells were in the module with a low percentage of wavelength-selective cells. From Zeki's Figure 8
, it is estimated that the color module and the orientation module were ~3 mm and ~4 mm wide respectively. Tanaka et al. showed that neurons with spectral or non-spectral properties were clustered within small, irregularly shaped patches 14 mm wide (Tanaka et al., 1986
). The sizes of these electrophysiologically defined modules are in the same range as the dense clusters in our model (Fig. 8
). Optical recordings have revealed V4 modules selective for color or orientation and large intervening areas containing cells with a diversity of receptive field properties (Ghose and Tso, 1995
, 1997
). These intervening areas, together with the large percentage of V4 cells with some degree of selectivity for wavelength or form, suggest a high degree of convergence between the two substreams in V4. As discussed before, the insertion by the secondary foci may play an important role in the convergence.
The modular afferent organization of V4 might have several functional correlates. First, the large size of the modules in V4 may be necessary to subserve aspects of color constancy that is compromised after lesions of V4 (Wild et al.,1985). In this way, comparisons of chromatic content can be made across large retinal distances within a V4 module and larger field comparisons can be made through module-specific connectivity. Second, psychophysicists have proposed the existence of different feature maps for different visual features such as color and form (Koch and Ullman, 1985
). The large modules in V4 may be part of the neural substrates of these feature maps. Since attentional effects were found in V4 (Moran and Desimone, 1985
), perhaps this organization allows for the switching of attention between color and form modules for rapid searching of objects based on one feature (i.e. red for ripe tomatoes). Responses of most V4 cells indeed depend on whether the stimulus was selected based on its color (Motter, 1994
).
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Acknowledgments |
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