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European Journul of Neuroscience, Vol. 1, pp. 494-506 @European Neuroscience Association 0953-81 &/89 $3.00

Modular Connections between Areas V2 and V4 of Macaque Monkey Visual Cortex

S. Zeki and S. Shipp Anatomy Department, University College London, London WC1 E 6BT, UK

Key words: cytochrome oxidase stripes, colour vision, functional specialization, integration

Abstract

We have studied the connections between two visual areas of macaque monkey cortex, V2 and V4, by injecting wheat-germ agglutinin horseradish peroxidase (HRP-WGA) into V4 and examining the distribution of labelled cells and terminals in V2, in relation to its characteristically striped cytochrome oxidase architecture. The cells projecting from V2 to V4 are arranged in bands and the number of bands per cycle of cytochrome oxidase stripes varies (one cycle consists of a thin stripe, a thick stripe and two interstripes). In the Type 1 connectivity pattern, there is just one band per cycle, centred over the thin stripes but normally spreading into the neighbouring interstripes. In the Type 2 connectivity pattern there are two bands per cycle, generally rather narrower and centred over the interstripes. Thick stripes are mostly free of labelled cells. The return projection from V4 to V2, whilst being concentrated in the vicinity of the labelled cells, is more diffusely distributed and invades the territory of all the stripes.

Introduction

For over one century, the clinical evidence purporting to show a colour centre in the cerebral cortex of man, outside the striate cortex, was repeatedly dismissed for a number of reasons (for reviews see Meadows, 1974; Zeki, 1989a). However, in 1973 the first evidence was obtained to show that separate areas of the visual cortex of the macaque monkey are involved in processing signals relating to colour (Zeki, 1973, 1977). The evidence was based on recordings from area V4 of the prestriate visual cortex. The presence of heavy concentrations of wavelength selective cells within this zone of cortex was in marked contrast to the physiology of an adjoining cortical area, V5, where most cells are directionally selective and none is wavelength selective (Zeki, 1974). These two studies led to a theory of functional specialization, according to which colour, motion, and form are processed in separate areas of the visual cortex (Zeki, 1978a). Furthermore the parallel outputs from V1 to these areas implied the presence of functional segregation within V1 itself (Zeki, 1975). Since then, it has been shown that this separation of functions is indeed a feature of the striate cortex (Livingstone and Hubel, 1984), a conclusion presaged many years earlier by both Wilbrand (1884) and Poppelreuter (1923), among others, on the basis of their clinical studies.

Yet V1 is not the only area which projects to the functionally specialized areas of the prestriate visual cortex. Another area, surrounding V1 and projecting to the very same areas, is V2 (Cragg, 1969; Zeki, 1969). Indeed, V4 was first defined on the basis of the input it receives from V2 (Zeki, 1971). V2 is a richly structured area with an internal modular organization also related to the separation

of functions. This modular organization was first shown by staining for the mitochondrial enzyme, cytochrome oxidase, which reveals two alternating sets of thick and thin darkly staining stripes, separated from each other by the interstripes which stain more lightly (Livingstone and Hubel, 1982; Tootell et al., 1983). Of particular interest here is the concentration of wavelength selective cells in the thin stripes, of directionally selective cells in the thick stripes, and of orientation selective cells in both thick stripes and interstripes (Shipp and Zeki, 1985; De Yoe and Van Essen, 1985; Hubel and Livingstone, 1987). Given the connections between V2 and V4, we wanted to learn whether there is a segregated connection between the two areas which reflects the specializations of the thin stripes of V2 and of area V4 for colour. We previously reported that both thin stripes and interstripes project to V4, and that the output to prestriate visual areas not involved in colour vision, namely V5 (MT) and V3, derives mainly from the thick stripes (Shipp and Zeki, 1985, 1989b; De Yoe and Van Essen, 1985). The work we describe here extends these results and goes on to consider the arrangement within V4 of the input from the thin stripes and interstripes, indicating that V4 itself is compartmentalized.

At the same time, we were curious about the distribution of the return projection from V4 to V2. Our studies of the connections between V2 and V5 showed that the return projection from V5 to V2 is much more 7 idespread than the outward projection, and not restricted to the territory of the thick stripes (Shipp and Zeki, 1989b). We wanted to learn whether such a widespread distribution is also characteristic of the return projection from V4 to V2 since such evidence might give

Correspondence to: S. Zeki and S. Shipp, Anatomy Department, University College London, London WClE 6BT, UK

Received 4 January 1989, revised 27 February 1989, accepted 12 April 1989

Connections of V2 and V4 495

us some insights into how the visual cortex achieves the remarkable feat of integrating the separately processed attributes of the visual scene to give us our unitary experience of the visual world.

Materials and Methods

Wheat germ agglutinin horseradish peroxidase (HRP-WGA), which is transported both retrogradely to label cell bodies, and orthogradely to label projecting fibres, was injected into cortical area V4 of eight monkeys anaesthetized with Sagatal and paralysed with Pavulon. Total quantities ranging from 0.5 to 0.05 pL of a 4% solution were injected by pressure using a 1 pL Hamilton syringe fitted with a glass micropipette tip. Three monkeys received multiple injections and the remainder one each. Following each injection, the syringe was left in the cortex for 10- 15 min and then rapidly withdrawn. If the tip was found to have blocked the procedure was repeated. After survival periods ranging from 48-72 h, the monkeys were given a lethal overdose of anaesthetic and perfused through the heart with 1 L of warm saline, followed by 3 L of buffered 4% paraformaldehyde and finally with sucrose solutions increasing in concentration from 10 to 30%.

Much of V2 is buried within the lunate (LS) and inferior occipital sulci (10s). The distribution of label within it is therefore best seen when the operculum is removed from the rest of the hemisphere, flattened and then sectioned parallel to the banks of the sulci (i.e. parallel to the plane of the paper, as shown in Fig. 1). This provides sections which pass through much of V2 in a plane parallel to its layering, though there are still parts of some sections where the plane of section is normal to the cortical surface. We cut sections at a thickness of 50 pm and stained alternate sections for HRP using tetramethyl benzidine (TMB) (Mesulam, 1982) and for cytochrome oxidase activity (Wong-Riley, 1979). Techniques for superimposing pairs of adjacent sections and reconstructing the relative distributions of the two kinds of label are described in Shipp and Zeki (1989a).

The remainder of the hemisphere was sectioned in our conventional near-horizontal plane. To compare the positions of the various injections in V4 we traced their outlines (the core region of heaviest deposition of TMB reaction product) from a 1 in 6 series of HRP-WGA sections and transferred the outlines onto a standard set of sections from a right hemisphere. The transfer was necessarily an approximate procedure since different brains vary somewhat in their sulcal patterns. The outlines of the sites were then plotted onto a surface view of the standard right hemisphere (Fig. 2). We estimate that the error in location of each site along the dorso-ventral axis is about f 1 mm.

Results

The present report is based on an examination of eight monkey hemispheres with HRP-WGA injections of varying size made into V4. Most injections were restricted to the posterior part of the V4 complex, lying in the anterior part of the lunate sulcus and the exposed posterior part of the prelunate gyrus (Zeki, 1971) (see Fig. 2). Deposits of HRP- WGA were found to extend from the site of entry of the micropipette into the cortex to the site of ejection, no more than 4 mm deep within the LS, producing an elliptical zone of uptake. In two hemispheres with multiple injections the injected area extended into the anterior part of the prelunate gyrus, thus involving the anterior part of the V4 complex, which receives a strong projection from the posterior part (Zeki, 1977). In terms of topography we expected, and found, labelled cells in the representation of lower visual field within the lunate and

W

FIG. 1. (Top) Diagram of the left hemisphere of a macaque monkey brain, with the occipital operculum reflected to reveal the parts of area V2 occupying the posterior banks of the lunate and inferior occipital sulci (LS and 10s). If the operculum is flattened and sectioned tangentially (i.e. in the plane of the paper) the sections pass roughly parallel to the layers of V2, in both the LS and 10s.

(Bottom) An operculum section stained for the enzyme cytochrome oxidase to reveal the characteristic striped architecture of V2. K = thick dark stripe; N = thin dark stripe; the pale stripes are known as interstripes (I). The distinction between thick and thin is easier for some stripes than for others; lower case letters indicate some degree of uncertainty in the identification in regions of irregularity.

parieto-occipital sulci, but not in the inferior occipital sulcus. The latter deals with the upper visual field, which past anatomical and physiological evidence has demonstrated not to be represented in the parts of V4 which we injected (Zeki, 1971; Van Essen and Zeki, 1978; Gattas et al., 1988).

Radial distribution of the label

Label distribution in different cortical layers was similar to that reported earlier (Rockland and Pandya, 1979; Lund et al., 1981; Fries and Zeki, 1983). All labelled cells in V2 were pyramidal and most were in the supragranular layers, mainly in the lower part of layer 3 (layer 3B). A few were present in layers 3A and 2, and also infragranularly, in layer 5 (Fig. 3). The layer 5 cells seemed to vary in frequency from hemisphere to hemisphere and were sometimes virtually absent-none, for example, is seen in Figure 3.

496 Connections of V2 and V4

FIG. 2. Drawings of the right hemisphere of a macaque monkey brain, with the occipital operculum partially removed to reveal the anterior bank of the h a t e sulcus, as viewed posteriorly (to the left of the figure) and laterally (to the right). The extent of the injection sites is shown separately in outline for each of the eight monkeys. Continuous outlines gave a Type 1 connectivity pattern in V2 whereas interrupted outlines gave a Type 2 connectivity pattern. No systematic relation between the sites of injection and the connectivity patterns is apparent.

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3

4

5

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FIG. 3. Dark field photomicrograph of a radial section (i.e. perpendicular to the layers) through one of the labelled patches in V2, following an injection of HRP-WGA into V4. Note the concentration of labelled cells just above layer 4, their absence in the lower layers, and the heavy distribution of terminal label in layer 1 and, to a lesser extent, in layers 5 and 6.

Scale bar equals 200 fim.

Labelled terminals, by contrast, were heaviest in layer 1 and much less heavy, but consistently present, in layers 2 and 3. They were lightest in layer 4 and became heavy again in layers 5 and 6 . This distribution is similar to that of orthograde tracers, such as tritiated amino acids, transported from V4 (e.g. Rockland and Pandya, 1979) so there is little doubt that it reveals the terminal arbors of the axons projecting from V4 to V2. The pattern is typical of a back projection from higher to lower areas, at least in the early parts of the visual cortex (Gilbert and Kelly, 1975; Tigges et al., 1977; Rockland and Pandya, 1979; Maunsell and Van Essen, 1983), and confirms the position of V2 as a lower area than V4 (Zeki, 1971). Some terminal label may also result from transport within the collateral axons of retrogradely labelled cells, though we note that these collaterals are principally located in layers 3 and 5 (Lund et al., 1981), and that there was relatively little sign of axonal label in layer 3 in our material. The tangential spreads of the orthograde and retrograde elements were not identical, a topic taken up below.

Tangential distribution of the label

The most prominent feature of the tangential architecture of V2 is a set of thick and thin cytochrome oxidase stripes, which are separated from each other by the less densely staining interstripes (see Fig. 1). In cytochrome oxidase stained material a single cycle, consisting of two interstripes, a thick stripe and a thin stripe, has a mean width of about 4 mm, ranging from 3.5 to 5 mm (see Shipp and Zeki, 1989b). As shown below, the projections from V2 to V4 are systematically related to this cytochrome oxidase architecture.

All of our injections yielded heavy groupings of labelled cells and terminals, ocurring at regular intervals and covering at least two cycles of cytochrome oxidase stripes. These groupings, which we refer to as bands, were oriented roughly orthogonal to the long axis of V2 and parallel to the cytochrome oxidase stripes. The bands had two different patterns of distribution: Type I, in which there was just one band per cycle of stripes, and Type 2, in which there were two.

Type I connections

Five of our eight injections produced this pattern, where the bands are centred on the thin stripes, but are sufficiently broad to invade the neighbouring interstripes on either side. They tend to avoid the thick stripes, but not always entirely so. Figure 4 shows an example in which the bands avoid the thick stripes, although examination at higher magnifications revealed a few labelled cells in them. Figure 5 shows a less regular example of a Type I connectivity pattern in which there may be a greater involvement of the thick stripes. For instance the thick stripe between the two central bands of label (each of which occupies thin stripes and interstripes) is spanned by a transverse band of cells, the whole giving rise to an ‘H’ shaped distribution of label. This is as clear an example of a segment of a thick stripe projecting to V4 as we have observed (though it may be significant that the part of the thick stripe involved is relatively low in cytochrome oxidase content). Two further thick stripes in this hemisphere may also be connected with V4 but in both cases there is a local anomaly which makes it difficult to determine the status of the labelled region as belonging unambiguously to a thick stripe. Thus the cytochrome oxidase content may be low, or the pattern irregular, involving fusion of adjacent thick and thin stripes, making it difficult to delineate each set (see legend to Fig. 5). There was a similar ambiguity involving one band in one other hemisphere with a Type 1 connectivity pattern.


FIG. 4. The distribution of HRP-WGA label in V2 (A and C), in relation to the pattern of cytochrome oxidase activity (B), following an injection into V4. The sections are from a flatmounted occipital operculum and are sequential, A being the most superficial. All three mainly pass through layers 3 and 4. K and broad arrows indicate thick stripes, N and slim arrows thin stripes. This is a Type 1 pattern, the four separate groups of labelled cells occupying two separate cycles of cytochrome oxidase stripes, assuming that the cluster of cells adjacent to the border with V1 (double arrowhead) belongs to the same stripe as the cluster of cells immediately above it. All the bands of labelled cells are centred on the thin stripes. The central band is the most clearly defined. It spreads into the interstripes on either side but does not extend as far as the two neighbouring thick stripes. To the left of it are two fainter and isolated cell clusters, near the surface (A) and near the white matter (C), which are at least partially coincident with the thick stripe. Also noteworthy is the continuous distribution of terminal label visible in layer 1 between the central and right-hand thin stripes.

Scale bar equals 2 mm.


FIG 5.


Type 2 connections

An example of a Type 2 connectivity pattern between V2 and V4 is shown in Figure 6, where the five bands of labelled cells are centered on the interstripes, giving two bands per cytochrome oxidase cycle. Small amounts of label extend between the bands across the thin stripes but the thin stripes are much less heavily involved than in the Type 1 pattern. The thick stripes are avoided by the bands. The Type 2 pattern was found in three brains, although in one of them the cytochrome oxidase sections were unsatisfactory. We nevertheless deduced from the breadth and spacing of the bands that this was very likely a Type 2 connection, the periodicity of bands in a Type 2 pattern being half that of a Type 1 pattern (see Fig. 7 and below).

It is evident from this that the great majority of the input to V4 from V2 is provided by the thin stripes and interstripes, and it would appear that some parts of V4 receive only from the interstripes whereas in other parts of V4 the contributions from thin stripes and interstripes are more equally matched. But we found no consistent relationship between the size and location of the injected part of V4 and the resulting connectivity pattern. Figure 2 shows that, as best we could determine it, a number of the Type 1 and Type 2 injection sites occupied equivalent locations in the different hemispheres. And, ranked in order of size, the Type 2 sites were smaller than the Type 1 sites, but not significantly so (p=O.3, Mann-Whitney test). Thus we obtained no clear indication of the size or disposition of the territories in V4 having a Type 1 or Type 2 connection with V2. But it may be telling that we observed no unequivocal example of a mixture of Type 1 and 2 connectivities in the same hemisphere, as if no injection involved both kinds of territory in V4. An explanation for this paradoxical observation is offered in the Discussion.

General characteristics of the bands

The bands themselves are made up of labelled cells, probably their axon collaterals and also the return projection from V4 to V2. The latter is heaviest immediately above and below regions of cell labelling. Therefore a band-like distribution is seen in all layers, though least clearly in layer 4 where the label is minimal. The distribution of labelled cells within the bands was not entirely uniform. Although individual sections might display only one or two clusters of labelled cells, superimposition of sections usually revealed a near-continuous distribution of labelled cells within the band, as shown in Figure 8. Heavier clusters of labelled cells within the bands did not seem to

correlate with the cytochrome oxidase sub-architecture of the thin stripes, which is commonly beaded. This differs from the distribution of the VS-efferent cells in the thick stripes, which shows a more obvious form of clustering, and partial correlation with the sub-architecture of the stripes (Shipp and Zeki, 1989b).

The bands in a Type 1 pattern were broader than the bands in a Type 2 pattern, although the general irregularities which are a feature of the bands made the determination of their exact widths awkward (see Figs. 4-6). Nevertheless, it was obvious that they are not of uniform thickness throughout their extent. In general, at their widest point, Type 2 bands ranged from 0.5 to 1.5 mm in width and Type 1 bands from 2 to 3 mm. The band periodicities also varied between the two types of connectivity pattern. For Type 1 the average periodicity was 4.0 mm, although it varied from 2.1 mm (Fig. 5) to 4.7 mm (Fig. 4). The mean periodicity of the Type 2 pattern (including the brain of Fig. 7) was 2.2 mm.

The return projection from V4 to V2

The most interesting feature of the return projection from V4 to V2, seen in all our brains, was its more widespread distribution, in the tangential plane, than the origin of the forward projection. Figure 9 shows the distribution of terminal label in a radial section passing through all layers. Although heaviest immediately above and below the labelled cells, it is continuously distributed in layers 5 and 6 and near-continuously in layer 1. It is best studied at higher powers of magnification, since at lower powers a relatively sparse distribution of terminal label may escape notice. There were occasional exceptions to this pattern in that the territory between some of the bands toward the edges of the labelled zone showed an absence of labelled terminals, in an unpredictable fashion.

The more diffuse distribution of the terminal label could also be seen in tangential sections through the lower layers, both in terms of the distribution of label between the bands and along their lengths (e.g. Fig. 6C). In general, the continuous distribution of labelled terminals contrasted with the patchy distribution of the labelled cells. Diffusely distributed terminal label usually extended a few mm beyond the outlying bands of labelled cells in the LS, and sometimes also into the IOS, where it was visible in layer 1. In one case, which yielded a Type 1 pattern in the LS, we observed sparse terminal labelling in layer 6 of the 10s which involved both thick stripes and thin stripes more than the interstripes. In another brain, the label in layer 1 was not restricted

FIG. 5. Photomicrographs to illustrate a Type 1 distribution of label in V2, following an injection into V4. Section A is the most superficial. Arrowheads point to some of the corresponding blood vessels in all three sections. K/N and broad/slim arrows indicate thick or thin stripes respectively but lower case letters and outline arrows are used where the identification of stripes is less certain. For instance, k2 becomes less distinct towards the top of the section and K3 towards the bottom; n3 and k4 are irregular and partly coalesce with each other-indeed the upper part of n3 might better be regarded as the left-hand branch of a bifurcation in K4; and n4 and k5, though more distinct, can only be identified according to the principle of alternation. All four bands of labelled cells and terminals involve mainly the thin stripes and interstripes, though each also forms spurs which cross into the territory of the thick stripes. Yet in each case this coincides with an indistinct or relatively lightly stained part of the thick stripe. Thus the bands centred loosely on N1 and N2 link diffusely across the top of k2 where k2 itself appears to fuse with its neighbours (A). The ‘H’ arrangement of the bands on N2 and n3 involves a lightly staining part of K3 (A). And the band on n4 extends a branch towards n3 passing through a gap in k4 no more heavily stained than the interstripes (C).

Scale bar equals 2 rnm.

FIG. 6. Illustration of a Type 2 distribution of HRP-WGA label in V2, following an injection into V4, using conventions defined in Figures 4 and 5. The five bands of labelled cells and terminals are situated in the interstripes, such that there are two bands of label per cycle of the cytochrome oxidase stripes. The thin stripes are much less heavily involved than in the Type I pattern, thus distinguishing the two patterns. The rightmost thick stripe and its neighbouring interstripes in B appear fainter than the others because this part of the section passes through layer 5 . The remainder of the section is in layers 4 and 3. Correspondingly, the rightmost pair of bands of label in C are represented mainly by labelled terminals alone. The leftmost band of label in C appears fainter still, presumably because it is here passing through layer 4 where the density of labelling is lightest.

Scale bar equals 2 nun.


FIG. 6.


FIG. 7. Dark field photomicrograph of a part of V2 taken from a flatmounted section through the occipital operculum and showing three bands of label following an injection of HRP-WGA into V4. The location of a fourth cluster of V4-efferent cells, somewhat sparsely labelled in this particular section, is indicated by an arrow head. Although adequate cytochrome oxidase sections were not obtained for this brain, the frequency of the bands is equivalent to that of the interstripes and their spacing suggests the presence of a thin stripe and a thick stripe in the unlabelled intervals. Both features are indicative of a Type 2 pattern of connectivity.

Scale bar equals 2 mm (adjusted to the typical level of shrinkage of cytochrome oxidase stained sections).

to V2 but continued well into V1 where it could be easily traced for some distance superficial to the familiar blobs.

In conclusion, it would seem that the back projection from V4 to V2 is more widely distributed than the origin of the forward projection. This reciprocal asymmetry is similar to the organization of the back projection from V5 to V2 (Shipp and Zeki, 1989b). since both return projections invade the territory of all three kinds of stripe.

Discussion

The present study forms an extension of previous work on the projections from area V2 to areas V3, V4, and V5 of macaque monkey prestriate cortex (Zeki, 1971; Shipp and Zeki, 1985). It amplifies earlier reports concerning the segregation of pathways through V2 and also suggests indirectly that area V4 too may be composed of distinct compartments. In addition we found that, like the connections of V2

FIG. 8. A computer-aided reconstruction of four HRP-WGA sections, showing bands of label in V2 following an injection into V4. The label appears as a small cluster in each individual section, which are from a 1 in 2 series (the intervening sections having been stained for cytochrome oxidase). But when the boundaries of the label in each section are superimposed on a single reconstruction, the bands appear to be continuous.

Scale bar equals 2 mm.

with V5, the return projections from V4 are not segregated to the same extent, which we can only suppose reflects some kind of role in tying together the processed output of the separate areas.

The colour and form pathways in the cerebral cortex

Taken in conjunction with the results of electrophysiological experiments (Shipp and Zeki, 1985; DeYoe and Van Essen, 1985; Hubel and Livingstone, 1987), the evidence presented here allows us to give a fairly detailed picture of the cortical pathways involving V4, a visual area of the prestriate cortex in which the vast majority of cells are wavelength selective, though many are also orientation or shape selective as well (Fig. 10). One pathway is derived from cells in the parvocellular (p) layers of the lateral geniculate nucleus, whose axons terminate in layer 4Cp (Hubel and Wiesel, 1972; Hendrickson et al., 1978). It then connects to the cytochrome oxidase blobs of layers 2 and 3 of V1 (Blasdel et al., 1985; Fitzpatrick et al., 1985), which in turn connect specifically with the thin cytochrome oxidase stripes of V2, zones in the two areas which are rich in wavelength selective and non-oriented cells which prefer low spatial frequencies (Livingstone and Hubel, 1984; Tootell et al., 1983, 1988a,c). Another input from the parvocellular laminae, through layer 4Cp, is to the interblobs of

FIG. 9. A high power, dark field view of a radial HRP-WGA section through V2 at the lip of the LS, taken from a flatmounted occipital operculum. This is the same brain illustrated in Figures 6 and 8, with bands of label principally occupying the interstripes (Type 2 pattern). Broad and slim arrows point to the locations of thick stripes and thin stripes respectively. Also visible here is the diffuse distribution of the terminal label, representing the return projection from V4 to V2, in areas between the bands of cells, in layers 1, 5 and 6. The dashed line indicates the junction with white matter.

Scale bar equals 1 mm (adjusted to the level of shrinkage of the accompanying cytochrome oxidase stained sections).


FIG. 10. A summary diagram of the colour and form pathways in the initial stages of the primate visual system (areas V 1, V2, V3 and V4) taken from the results reported here and elsewhere. Input to layers 2 and 3 of V 1 is ultimately derived from the parvocellular (P) layers of the lateral geniculate nucleus and that to layer 4B from the magnocellular (M) layers. But the output of layers 2 and 3 has two distinct components, arising from the blobs and interblobs, respectively carrying information about the spectral and spatial attributes of the image. The course of the three forward pathways from V 1, originating in layer 4B, the blobs and the interblobs, are indicated by broad, medium and slim arrows respectively. Layer 4B projects to V3 directly and via the thick stripes of V2, as indicated. The pathways from layers 2 and 3 both lead to V4, the blobs via the thin stripes and the interblobs via the interstripes. Both V3 and V4 are involved in the analysis of form, and V4 is also involved in the analysis of colour. Although V3 and V4 are interconnected, and though there may be additional direct contributions of the M system to V4 (see text), it is generally true that the form and colour components of the P system retain a closer association with each other than either develops with the form component of the M system. But the precise organization of the colour and form pathways within V4 remains to be determined. We have indicated that V4 may contain Type 1 and Type 2 compartments. Type 2 receives input from the interstripes alone; Type 1 receives input from the thin stripes and possibly also from the interstripes.

VI, which project to the interstripes of V2 (Livingstone and Hubel, 1984), the two zones containing abundant orientation selective cells, but fewer wavelength selective ones (Shipp and Zeki, 1985; DeYoe and Van Essen, 1985; Hubel and Livingstone, 1987). Both thin stripes and interstripes project to V4. From V4, separate pathways lead to the parietal and inferior temporal cortex, (Zeki, 1977; Desimone et al., 1980; Seltzer and Pandya, 1980; and authors’ unpublished results) although the former projection may be limited to extrafoveal regions of V4 (authors’ unpublished results). How the output of V4 to these two regions of the cortex is arranged with respect to the input they receive from other visual areas has yet to be uncovered.

It is important to emphasize that the magnocellular (M) system, which runs from the magnocellular layers of the lateral geniculate nucleus and is relayed through layer 4B of VI to V3, both directly and through the thick stripes, also carries form information, though probably of a different nature (Shipp and Zeki, 1985; Burkalter et al., 1986; Livingstone and Hubel, 1987a,b; Zeki and Shipp, 1988). Our results suggest that V4 itself receives a small contribution from the magnocellular system, via the thick stripes, and a magnocellular contribution to the blobs of V1 has already been suspected (Tootell et al., 1988b). But, in general, the form and colour components of the P system appear to retain a closer association with each other than either develops with the form component of the M system. This leads us to consider (A) whether there is any compartmentalization within V4 for the form and colour pathways projecting to it; (B) the psychophysical evidence for the kind of functional activities that are facilitated by the continued association of the form and colour pathways in a single area; and (C) the clinical evidence regarding this association.

(A) Does V4 have a modular organization?

Our injections into V4 produced two distinctly different patterns of labelled cells in V2 (see Fig. I l ) , as if the injections had been placed within different compartments, with different patterns of connectivity. Provisionally, we can identify the compartments on this basis: a Type 1 compartment in V4 has Type 1 connections with V2 and a Type 2 compartment has Type 2 connections. Thus the Type 2 compartment would receive input solely from the interstripes and the Type 1 compartment from both thin stripes and interstripes. Yet no brain displayed the mixture of Type 1 and Type 2 connections that we might have expected from an injection straddling the border between compartments, suggesting another possibility: if the Type 1 compartments were smaller than the Type 2 and occupied a smaller proportion of V4 (perhaps by occupying islands within a matrix of the Type 2 compartment), it is possible that none of our injection sites fell into a Type 1 Compartment without also involving a Type 2. If this were the case, we can envisage that the Type 1 compartment might receive input solely from the thin stripes, despite the fact that we never observed such a limited distribution of labelled cells in V2.

In either case, the anatomical evidence implies two sets of repeated compartments within V4, each of which probably contains physiologically distinct populations of cells. Indeed, earlier physiological evidence (Zeki, 1983a) has hinted at such compartmentalization by revealing regional variations in the prevalence of wavelength and orientation selectivity. In addition to these properties, V4 cells have also been reported to vary in their selectivity for the length or breadth of a bar stimulus, and in their tuning for spatial frequency (Desimone and Schein, 1987). There are thus a number of ways in which the properties of the two compartments could differ. But the great majority of V4 cells, including those with form selective properties, are also wavelength selective or biased (Zeki, 1973, 1978; Desimone and Schein, 1987). How wavelength selectivity is generated in Type 2 compartments, which receive their inputs from the interstripes, where most cells are orientation but not wavelength selective, is not clear. One possibility is that the interstripes have substantial numbers of wavelength selective cells, as was indeed reported by DeYoe and Van Essen (1985). A second is that minor wavelength biases in interstripe cells may be amplified by local interactions within V4. Finally, intrinsic lateral connections with Type 1 compartments may confer this property on the Type 2 compartments by means of some kind of gating


FIG. 1 1. A schematic diagram of the two types of connectivity pattern between V2 and V4. In Type 1 (above), there is one band of V4-efferent cells per cycle of cytochrome oxidase stripes, centred on the thin stripes. In Type 2 (below), there are two bands, centred on the interstripes.

mechanism. Such intrinsic connections are fairly extensive in V4 (Zeki, unpublished results).

(B) The psychophysical evidence for the relationship between form and colour information

The interplay between form and colour may occur in both directions: spectral information contained in the image may play a role in determining form, and spatial information might play some role in perceiving colour.

(i) The detection of boundaries is of fundamental importance in colour vision. Krauskopf‘s experiments showed that if the boundary between two colours is made to disappear by image stabilization, then one colour also disappears and is replaced by the other (Krauskopf, 1961). It is as if different colours are only seen by virtue of the contrast at their borders and that information arriving from within the bcdy of the colour is discarded. Land’s Retinex Theory (Land and McCann, 1971; Land, 1974), derived from his psychophysical experiments, postulated just such a mechanism in trying to account for colour constancy (the fact that surface colours are largely independent of the nature of the prevailing illumination). Borders between colours are detected by comparing the light reflected from neighbouring points. If great, the difference is assumed to be due to the points belonging to surfaces with different reflectances; if small, the differences are attributed to uneven illumination and ignored. The Craik-Cornsweet -0’Brien illusion (in which two surfaces of identical reflectance appear to differ in lightness-Cornsweet, 1970) confirms that the visual system does indeed place more weight upon sharp changes than gradual ones in determining the lightness of an area from information obtained at its border. Though not quite as strong as the achromatic version, the Cornsweet effect can also be demonstrated using isoluminant chromatic

gradients (Ware and Cowan, 1983). This indicates that the chromatic pathways themselves are capable of detecting boundaries and computing lightnesses within limited spectral bandwidths, a possible function of some V4 cells (Zeki, 1983b). V4 is also well-equipped to perform the final integration of the difference signals, allowing the diffuse filling- in of colour within a boundary, by virtue of the spatially convergent projections it receives from V2 (Zeki, 1971) and its extensive intrinsic connections (Zeki, unpublished results).

Whether the processes described above of necessity require oriented wavelength selective cells is not clear. The two-dimensional algorithm of Horn (1974) for computing lightness is formulated in terms of centre- surround operations only (the rotationally symmetric Laplacian and inverse-Laplacian operators), though Blake (1985) improved it by replacing the initial Laplacian with an oriented gradient operator. Thus theoretical considerations suggest that such operations may be undertaken by oriented, or non-oriented, cells. Perhaps the clearest account of how orientation selective cells might contribute to this process has been developed by Grossberg (1987). This model indicates how competitive and cooperative interactions between orientation selective cells can restore a distinct object boundary, to be diffusely filled in by a particular colour, overcoming the image degradation inherent in the optics of the eye. Though the model is principally formulated with respect to V1, V4 might be involved by means of feedback loops, or it might perform a similar and independent process involving interactions over a wider part of the field of view.

Finally we should note that the lightness algorithms mentioned above are competent only for two-dimensional surfaces. The shading and shadowing due to real three-dimensional objects pose additional obstacles for colour constancy. How shading, and other information about depth, might interact with the mechanisms responsible for constancy, and whether orientation selective cells are essential for such a process, remains unknown.


(ii) In the reverse direction, the contribution of chromatic or spectral information to form vision is evident in the fact that simple forms can be distinguished in isoluminant stimuli, even if the image takes on a ‘jazzy’ appearance (Gregory, 1977). We assume that this task is initiated in the interblobs of V1, by oriented complex cells which can respond to contours defined by isoluminant combinations of lights of different wavelength, though the cells are not themselves wavelength selective (Gouras and Kruger, 1979; Thorell et al . , 1984). The question that remains to be answered is why this output from the interblobs is not separated and processed separately but routed to the same area as that which may be using spectral information to compute surface reflectance (i.e. to achieve colour constancy), namely V4.

V4 may thus be involved in several tasks for which both colour and form information is necessary, depending on both topical and confluent convergent cortical connections (Zeki and Shipp, 1988). These may be summarized as follows: the construction of colours, entailing lightness computations within three spectrally different channels; matching the boundaries of objects and surfaces within these three channels, a process in which achromatic contours may be dominant (border locking); and the use of colour and lightness information in the determination of forms, possibly in three dimensions. V4 may also employ information derived from the M system in determining form, perhaps in association with V3 with which it is interconnected (authors’ unpublished results).

(C) The clinical evidence for the separation of form and colour

This very association between form and colour raises problems of interpretation, mostly because of the clinical evidence. It is now well established that, following lesions in the prestriate cortex, and more specifically in the lingual and fusiform gyri, colour vision can be compromised. In the majority of such achromatopsic patients there is a scotoma and some other associated deficit, commonly an inability to recognize familiar faces (prosopagnosia). The scotoma is due to involvement of the calcarine cortex and the prosopagnosia to involvement of a neighbouring, specialized, region of the prestriate cortex. Commonly, too, there has been a diminution of visual acuity. What is important to emphasize, however, is that there are at least five reported cases of cerebral achromatopsia which are not associated with a scotoma, a diminution of visual acuity, a prosopagnosia or a defect in form perception (unpublished observation). The most recent of these have been reported by Kolmel (1988) and by Sacks et al. (1988), the latter a patient one of us (SZ) had the opportunity to examine. These are cases of pure achromatopsia, and in the patients of Kolmel, at least, are associated with occipital lesions outside the striate cortex, as determined from brain scans.

This clinical evidence appears to contradict the experimental evidence which suggests a more intimate relationship between colour and form in area V4 of the macaque monkey. One interpretation of this discrepancy is that an area of the human prestriate cortex has become uniquely involved with colour vision. If so, it becomes necessary to suppose that the form information necessary for generating colours in that area is duplicated elsewhere. An alternative interpretation is that there is, in the human brain, a visual area functionally identical to macaque monkey V4 but that achromatopsia results when the blood supply of the occipital lobe is partially disrupted, either through a vascular accident or a lesion. This process may affect the metabolically more active blobs more severely with the consequence that the entire colour pathway is specifically compromised. Such an explanation

accounts for the ascotomatous achromatopsia observed in some patients, for example that of Steffan (1881), Kolmel (1988) and Sacks et al. (1988).

Experimental studies in the macaque have not resolved this difficulty. Wild et al. (1985) described two monkeys with lesions in V4 which were more impaired in a colour constancy task than either wavelength or form discrimination tasks. Heywood and Cowey (1987) did not employ a colour constancy task but did find significant impairment of both form and wavelength discrimination. This difference might have resulted from making the cortical lesions more extensive. Future studies should reveal the exact nature of the defects to be expected from lesions of V4 and whether there is a substantial difference between the effects of such lesions and those produced in the lingual and fusiform gyri in man.

The reintegration of form, colour and motion

In the past, we have emphasized the separation of form, colour, and motion. Anatomical and physiological evidence and, more recently, psychophysical and novel clinical studies, speak strongly in its favour. Separate specialized anatomical pathways within areas VI and V2, whose existence was not even suspected before the discovery of functional specialization in the prestriate visual cortex, have now been charted in some considerable detail. Taken together, these results provide at least a partial explanation for the capacity of the visual system to process different attributes of the visual scene separately. Yet perhaps the greatest perceptual experiment of all is the one that we each experience continually during our normal lives, namely that the various attributes of the visual scene are seen in exact spatio-temporal registration. Although the study of this subject is still in its infancy, anatomical evidence provides a strong hint that it occurs at different levels, including levels at which functional specialization is first established in the visual cortex, that is at the levels of V1 and V2 (Zeki and Shipp, 1988). In this paper, we have shown that all the functionally segregated stripes of V2 receive a return projection from V4, just as all the stripes also receive a return input from V5 (Shipp and Zeki, 1989b). Why it is that integration may occur at such an early level is a topic we have addressed elsewhere (Shipp and Zeki, 1989b; Zeki, 1989). Among the important reasons may be that as one proceeds to areas such as V4 or V5, the increase in receptive field size renders more difficult the task of identifying whether simultaneous activity in the two areas is due to the same object. Perhaps the reciprocal connections with V2, where topographic precision is better preserved, help to facilitate this process.

There are, of course, other pathways for bringing about the integration of the different visual attributes (Zeki and Shipp, 1988) and the role of the reciprocal projections in relation to these other pathways remains to be understood. But the anatomical evidence that we have presented here and elsewhere highlights, at the very least, the stages at which one might search physiologically for the processes underlying integration.

Acknowledgements

The credit for the histology belongs to Ian Wilson. The authors also thank Dr Phillip Cope for assistance with computer graphics.

This work was supported by grants from the Wellcome Trust and from the Science and Engineering Research Council.


Abbreviations

HRP-WGA I 10s K LS M N P TMB

wheatgerm agglutinin-horseradish peroxidase interstripe inferior occipital sulcus thick cytochrome oxidase stripe lunate sulcus magnocellular thin cytochrome oxidase stripe parvocellular tetramethyl benzidine

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