Functional Demarcation of a Border Between Areas V6 and V6A in the Superior Parietal Gyrus of the Macaque Monkey

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1 European Journal of Neuroscience, Vol. 8, pp , European Neuroscience Association Functional Demarcation of a Border Between Areas V6 and V6A in the Superior Parietal Gyrus of the Macaque Monkey C. Galletti, P. Fattori, P. P. Battaglini ~~, S. Shipp2 and S. Zeki2 Cattedra di Fisiologia generale, lstituto di Fisiologia umana dell Universita di Bologna, Piazza di Porta S. Donato 2, Bologna, Italy 2Department of Anatomy, University College, Gower Street, London WCl E 6BT, UK 3Present address: lstituto di Fisiologia dell universita di Trieste, Via Fleming 22, Trieste, Italy Keywords: parietal cortex, area PO, laminar connections, functional maps, functional streams Abstract We have compared physiological data recorded from three alert macaque monkeys with separate observations of local connectivity, to locate and characterize the functional border between two related but distinct visual areas on the caudal face of the superior parietal gyrus. We refer to these areas as V6 and V6A. They occupy almost the entire extent of the anterior bank of the parieto-occipital sulcus, V6A being the more dorsal. These two areas are strongly interconnected. Anatomically, we have defined the border as the point at which labelled axon terminals first adopt a recognizably descending pattern in their laminar characteristics, after injections of wheatgerm agglutinin-horseradish peroxidase into the dorsal half of the gyrus (in presumptive V6A). A similar principle was used to recognize the same border by the pattern of input from area V5, except that in this case the relevant transition in laminar characteristics is that between an intermediate pattern (in V6) and an ascending pattern (in V6A). V6A was found to be distinct from V6 in a number of its physiological properties. Unlike V6, it contains visually unresponsive cells as well as units with craniotopic receptive fields ( real-position cells), units tuned to very slow stimulus speeds, units with complex visual selectivities and units with activity related to attention. V6A was also found to have a larger mean receptive field size and scatter than V6. By contrast, response properties related to the basic orientation and direction of moving bar stimuli were indistinguishable between V6 and V6A, as was the influence of gaze direction on cell activity in the two areas. Two-dimensional maps of the recording sites allowed reconstruction of the V6N6A border. For comparison, the anatomical results were rendered on two-dimensional maps of identical format to those used to summarize the physiological data. After normalizing for relative size, the physiological and connectional estimates of the border between V6 and V6A were found to coincide, at least within the range of individual variation between hemispheres. An architectonic map in the same format was also made from a hemisphere stained for myelin and Nissl substance. Area PO, defined by its general density of myelination was not distinct in this material, but several architectural features were traceable and one of these was also found to approximate the V6N6A border. The particular criteria that distinguish V6 from V6A differ from a recent description of areas PO and Pod in the Cebus monkey; we believe it most likely that PO and Pod together may correspond to V6. introduction The delineation of the borders between neighbouring areas in the prestriate cortex of the monkey is often a gradual process, one that is dependent on the progressive combination of a number of diverse criteria. In this report we combine anatomical and physiological data from our two laboratories in order to analyse the functional organization of the caudal face of the superior parietal gyrus (SPG), and in particular to chart the border between two functionally distinct visual areas which we shall refer to as V6 and V6A. Visual responses on the caudal face of the SPG4.e. the anterior bank of the parieto-occipital sulcus (P0S)-were first described by Gattass and his colleagues (Covey et al., 1982; Gattass el al., 1985). who designated this new visual area PO. The definition was based on a combination of myeloarchitectural and visual receptive field mapping criteria. In subsequent reports from the same group (Colby ef al., 1988; Neuenschwander et al., 1994), the field PO has progressively contracted in size, owing to reassessments of the above criteria that have shifted the dorsal border of PO downward along the anterior bank of POS. Thus, according to the present definition, area PO is a small, ventral part of the territory originally given the name PO. The term V6 was introduced by Zeki (1986) to describe a visual Correspondence to: Prof. C. Galletti, as above Received 17 January 1995, revised 19 June 1995, accepted 28 July 1995

2 Areas V6 and V6A in the SPG of macaque 31 region in the anterior bank of the POS that was circumscribed by a ring of callosal connections, on the hypothesis that the callosal pattern described the border of the area, as it does for areas V3 and V3A in the posterior bank of the POS itself. Later work has provided anatomical and physiological substantiation of the callosal border, if also revealing that the callosal connectivity of the region is more complex and variable than first thought (Shipp and Zeki, 1987; S. Shipp, M. Blanton and S. Zeki, manuscript in preparation). Overall, the evidence so far is sufficient to confirm that this region of cortex is indeed visually active and distinct from visual areas such as V2, V3 and V3A lying ventrally and laterally, or the part of area 5 to be found more anteriorly within the neighbouring part of the intraparietal sulcus. Nonetheless, the exact borders of callosal V6 are difficult to pinpoint, as are the borders of area PO, however formulated-and the perimeters of the two areas have yet to be clearly delineated or shown to correspond. In the present paper we report evidence to pinpoint the location of an internal border within the ambit of callosal V6. This border bisects callosal V6 into dorsal and ventral regions with different anatomical and functional characteristics. An analogous distinction was initially made by Colby et al. (1988). Their dorsal region (defined by light myelination) had visual neurons that were very difficult to drive and with receptive fields much larger than those of the ventral region (their area PO). Later on, Galletti et al. (1991a), recording from the caudal pole of the SPG in alert behaving monkeys, found a dorso-medial region containing a mixture of visual and non-visual cells, in contrast to a ventro-lateral region that was purely visual. This seemed to reflect a dissimilarity in the arrangement of extrinsic connections, for the more ventral cortex receives stronger inputs from other regions of visual prestriate cortex such as V2 and V3A, and the more dorsal cortex has closer relations with somatosensory and motor fields, such as parietal area 5PE, and area 6 of frontal cortex; the ventral and dorsal regions are also strongly connected to each other (S. Shipp, M. Blanton and S. Zeki, manuscript in preparation). The pathways from the visual cortex on the caudal pole of the SPG to regions of somatosensory association cortex and premotor cortex are indicative of the role these areas must play in visuomotor coordination. Further insight into the functional properties that sustain this role has been provided by the recordings from the SPG of alert monkeys: the visual responses of many neurons in this region are found to be regulated by extraretinal factors such as the direction of gaze or the nature of the behavioural task that the animal performs (Galletti et al., 1991a). Among these is a small proportion whose receptive field is expressed in head-centred coordinates and is thus ihvariant with respect to eye position (Galletti et al., 1993). Properties of a similar kind are also reported to be present in the frontal regions with which callosal V6 communicates (Gentilucci et al., 1983; Fogassi et al., 1992). Our working hypothesis has been that the evolution of more complex neuronal response properties in this region of the SPG parallels the transition from the purely visual properties of prestriate cortex to the visual-somatic associative areas of the parietal lobe. Accordingly, we have evaluated the visual and extraretinal properties of all the neurons so far recorded from the SPG for characteristics that might signal the distinction between a higher and lower area in a serial pathway. The locations of all these neurons were mapped on reconstructions of the posterior face of the SPG, in order to reveal any possible spatial segregation. This analysis indeed suggests the presence of a dividing line, the prospective border between V6 and V6A, which is angled obliquely across the face of the SPG. Anatomical data, in which the same provisional border has been identified by local changes in the pattern of laminar connections, were recast into the same format that was used for the physiological analysis. The structural and functional data sets, expressed as two-dimensional maps based on parasagittal sections, were found to be in close correspondence on the location of the border, lending confidence to the demarcation of areas V6 and V6A as separate and distinct units of cortical processing. Some preliminary data have been previously published in abstract form (Galletti et al., 1991b). Materials and methods Physiological recordings The technical details of our recording procedure are described by Galletti et al. (1984, 1995). Briefly, three juvenile monkeys (Macaca fascicularis) were trained to look at a fixation spot for 2-6 s without reacting to any other visual stimuli during this period. Stimuli were light or dark spots, or bars of variable dimensions and orientations, which were flashed onto or moved over a translucent screen (80 X 80 at 57 cm) by rear projection using an optical line. By moving the fixation spot to different locations on the screen, receptive fields up to -70 in eccentricity could be plotted in all four visual quadrants. Some visual cells were unresponsive to these stimuli, and could only be activated by more elaborate procedures involving manipulations along the optical line to generate shadows on the screen with complex forms and movements. The effect of changing the animal s direction of gaze upon a cell s activity was tested in a way fully described in Galletti et al. (1993, 1995). Briefly, the fixation spot was presented at nine different screen locations, at each of which the receptive field was replotted using the same stimulus. The animal s direction of gaze and the neural response level were continually monitored, in order to check for any gazedependent regulation of neural discharge, or any departure from a strictly retinotopic organization of the neuron s receptive field. Overall, we recorded from four hemispheres (three left and one right) in the three animals. During the last 2 weeks of recording from an animal, electrolytic lesions (30 ya cathodal current for 30 s) were made at specified coordinates within the recording chamber and, under barbiturate anaesthesia immediately preceding termination, the brain was stabbed with pins to mark fixed points according to the same coordinate system. Perfusion was carried out with warm heparinized saline, followed by 4% buffered paraformaldehyde and, in one animal, by sucrose in stepped concentrations from 10 to 30%. Brains were removed, photographed and sectioned parasagittally at pm thickness and 1-in-4 serial sections were stained with cresyl violet. In three hemispheres, alternate sections were stained for myelin using the silver method of Gallyas (1979). Electrode tracks with lesions, and some of the penultimate penetrations, were directly identified; the positions of the remainder were interpolated within the chamber coordinate system according to the specific points marked by the lesions and pinholes. The position of each penetration was then confirmed or slightly modified, if necessary, by reference to other factors, such as the depth profile of the penetration (the sequence and lengths of traverses through grey and white matter) and the retinotopic site of passage through the parts of V1 and V2 overlying the recording site in V6 complex. The approximate location of each recording site along the track was determined by its distance from the surface of the hemisphere and from other landmarks such as marking lesions, boundaries between white and grey matter and, especially, the point of crossing through the POS.

3 32 Areas V6 and V6A in the SPG of macaque Anterior bank of Pos W FundusofPOs Medial bank of Ips II Occipital lobe Dorsal surface of the superior parietal gym Medial surface of the hemisphere E M Ventral surface of the hemisphere 2 3 FIG. 1. Bidimensional map of the caudal pole of the superior parietal gyrus (SPG) illustrating the method used to chart the relative locations of recording sites. Three parasagittal sections of the posterior half of the brain, passing through the caudal SPG, are shown on the top of the figure. At the bottom left is a brain model with the intraparietal, lunate and parieto-occipital sulci (IPS, Ls and POs) open, to reveal the full extent of the caudal pole of SPG. Different regions of the caudal SPG are distinguished by different patterns of shading in the sections and model, and in the two-dimensional map at bottom right. The twodimensional map has been constructed by partial unfolding of the contours of layer 4 from serial sections at 300 pm intervals, according to the method described by Van Essen and Zeki (1978). The thicker lines labelled 1, 2 and 3 in the two-dimensional map correspond to the dense contours (layer 4) from the three parasagittal sections illustrated above. An example of a reconstructed microelectrode penetration with three recording sites in the grey matter of the anterior bank of POs is shown in section 2. Although the recording sites were actually in the supragranular layers, they are radially projected onto layer 4 within the plane of the section in order to determine their position in the two-dimensional map. The square dashed outline indicates the part of the two-dimensional map that is shown in the following figures. Note that it is centred on the anterior bank of the POs (light grey area).

4 Areas V6 and V6A in the SPG of macaque 33 Two-dimensional reconstructions of the recording sites We used the partial unfolding method of Van Essen and Zeki (1978) to construct bidimensional maps of the posterior pole of the SPG. Figure I shows the correspondence between brain regions on the parasagittal sections and the same regions in the bidimensional map. Essentially, the construction of the map depends on two fixed points of reference in parasagittal sections. One of these is the crown of the SPG, the dividing line between the cortex in the anterior bank of the POS and that on the dorsal surface of the hemisphere (the line between the dark- and light-grey areas in Fig. 1). On the twodimensional map this line follows an oblique curve that exactly matches its profile in the intact brain. The other fixed point of reference on parasagittal sections was the dividing line between the anterior bank of POS and the medial surface of the hemisphere (the line between light-grey and dashed areas in Fig. 1). To afford comparability between different cases these two reference lines adopted the same position in all our two-dimensional reconstructions. Elsewhere the forms of the contours derived from individual sections were free to reflect the variable gyral morphology of each case. Because the posterior face of the SPG approximates to a flat surface, the reconstruction was considerably simplified by arranging the contours through this face parallel to each other and to the medial reference line (light-grey area in Fig. I). We introduced three discontinuities between the surrounding cortical surfaces, at points of maximum local curvature. One is at the foot of the SPG, where the contours can curve either onto the ventro-medial surface, or link posteriorly with the occipital lobe, depending on the case in question (oblique linedcross-hatched areas in Fig. I). The other two discontinuities follow the margins of the dorsal surface of the SPG: medially, at the junction with the medial surface of the hemisphere (dark-grey/ dashed areas in Fig. 1); and laterally, at the junction with the medial bank of the intraparietal sulcus (dark grey/dark-dotted areas in Fig. 1). The recording sites from each case were marked on the twodimensional map according to our best estimate of the locations of each electrode track and of the cells along the tracks. The maps and recording sites were digitized, and linked by purpose-written software to the database storing the functional properties of each unit, enabling rapid analysis of the distribution of any given functional characteristic. The maps of all four cases were subsequently combined to give an overall summary diagram. To do this they were aligned according to the two reference lines described above. As we had elected to standardize on the left hemisphere, the one map derived from a right hemisphere was reflected. Three hemispheres were very similar in size and their two-dimensional maps were accordingly similar in scale; the fourth (a left hemisphere) was smaller and we decided to rescale its two-dimensional map proportionally before superimposing it on the others. The rescaling factor (18%) was estimated from the relative size of the whole brain and from the dorso-ventral extent of the SPG, from its crown to its foot. Anatomical procedures The anatomical results reported here are based on injections of wheatgerm agglutinin-horseradish peroxidase (WGA-HRP; Sigma; pl of a 4% solution in H20) into the visual cortex of four macaque monkeys (Macacafascicularis). Three animals were injected in the caudal pole of the SPG, two in the left and one in the right hemisphere (S. Shipp, M. Blanton and S. Zeki, manuscript in preparation). The fourth was injected in the caudal superior temporal sulcus in the left hemisphere; this injection was centred in area V5, identified by prior recording as described by Shipp and Zeki (1989). All brains were sectioned horizontally at pm. Every third section was reacted for WGA-HFW according to the methods of Mesulam (1982), and alternate sections were counterstained with cresyl violet. A set of sections from two hemispheres was FIG. 2. The generation of parasagittal sections by reslicing a three-dimensional reconstruction. (A) A parasagittal slice generated by reslicing (anterior is to the right, dorsal up); each broken horizontal line corresponds to one of the original horizontal sections, the breaks in the lines to sulci. (B) Contour line through layer 4 of the horizontal section corresponding to the bold line in A (anterior is to the right, lateral up); the horizontal line in B shows the location of parasagittal section A. (C) Occipital view of a three-dimensional reconstruction, partially dissected as shown in Figure 31 to reveal the anterior banks of the parieto-occipital and intraparietal sulci. In this case, an injection of WGA-HRP into area V5 (injection site not visible) was made; brightened segments of contour lines indicate the density of transported label. The single, uniformly bold contour line corresponds to horizontal section B; the vertical line gives the location of parasagittal slice A.

5 34 Areas V6 and V6A in the SPG of macaque saved and subsequently stained for myelin; this material was of indifferent quality. The HRP sections were examined under bright-field and crosspolarizing optics, the latter with a view to identifying different laminar patterns of labelled axonal terminals, and hence the hierarchical rank of the connected cortex with respect to the site of injection (Felleman and Van Essen, 1991). Patches of label were thus classed as higher and lower (terminal label concentrated within layers 4 and 1 respectively) or as intermediate (a more even distribution of terminal label). The classification of each patch of label was based on several sections, especially at sites where the local plane of section intersected radial columns of label at an oblique angle. Additionally, to substantiate our analysis of cortical architecture, one hemisphere from a fifth animal with no injected tracer was cut parasagittally and two adjacent sets of sections were stained for myelin (Gallyas, 1979) and Nissl substance (cresyl violet) respectively. Reconstruction of anatomical material The starting point for this analysis was a series of three-dimensional reconstructions which will be described in more detail elsewhere (S. Shipp, M. Blanton and S. Zeki, manuscript in preparation). Briefly, layer 4 is traced from each section, and registered in three-dimensional space by purpose-written software according to multiple cortical landmarks. The density of labelling (cellular and/or terminal) is recorded in grades from 0 to 4, and can be a summation of all layers, or of any individual layer projected radially onto layer 4. Examples of both types are shown here. Although the three-dimensional images have a higher topological fidelity than the two-dimensional maps, they are less lucid for making comparisons between individuals with variant gyral morphologies. Because the three-dimensional reconstructions are derived from horizontal sections, a method was developed for reslicing them in the parasagittal plane (Fig. 2) to yield outlines of layer 4 which were submitted to the same procedure that was used to generate twodimensional maps of physiological function. We reasoned that constructing both the anatomical and physiological two-dimensional maps from a parasagittal basis would enhance the accuracy of superimposition by reference to equivalent morphological landmarks, and minimize the scope for systematic differences in map construction. Conversely, two hemispheres with physiological data were also digitized as three-dimensional reconstructions (not illustrated here); by this combination of reconstruction techniques we achieved a reliable level of cross-reference between anatomical and physiological data that had initially been obtained in different planes of section. Statistical analysis We used standard techniques of linear regression to examine the relationship of receptive field size and receptive field scatter to eccentricity within the visual field, and analysis of covariance (ANCOVA) to test whether these relationships differ between the dorsal and ventral parts of the caudal SPG (V6 and V6A). In general, ANCOVA establishes whether two or more regression lines differ significantly in size and/or slope by evaluating alternative models of the data. For instance, in Figure 19 the receptive field data have been subdivided into two groups, described by two regression lines of identical slope. To test whether the difference in of these lines is significant an F-test is used to compare this model to a simpler one, in which all of the receptive field data from both groups are described by a single regression line. If the outcome is positive, a third model can be considered, in which the regression coefficient (slope) of each data set is computed independently, in order to determine whether the difference in slope is significant. All the data points admitted to this analysis were averages of the receptive field sizes or scatters recorded in a single penetration. Penetrations with fewer than four visual units were discarded. This is a conservative approach, but it avoids the lack of statistical independence inherent in analysing receptive fields on a unit-by-unit basis, since nearby units are likely to have correlated receptive field properties. Receptive field scatter was defined as the displacement of a receptive field centre from its predicted position, assuming a linear transect of the visual field as the electrode is pushed through the cortex. The latter is estimated by regressing the x and y coordinates of the receptive field centre against distance along the electrode track, as measured by the depth readings from the microdrive apparatus. The overall scatter between the number of units recorded in a single penetration was computed as a root mean square (RMS) value: RMS scatter (single penetration) = [root](z square displacementh - 2) (n - 2) replacing n as the denominator to compensate for the tendency of penetrations with few receptive fields to yield anomalously low values of scatter. Results Anatomical demarcation of the V6N6A border Determination by laminar patterns of connectivity In principle, the border between the ventral and dorsal areas on the caudal SPG that we identify as V6 and V6A should be recognizable by a localized change in the laminar organization of cortico-cortical connections, wherever bands of WGA-HRP labelling cross between the two areas. The examples we describe here are drawn from four hemispheres, one with an injection into V5 (Shipp and Zeki, 1989) and three with injections into the dorsal part of V6 complex (defined by its perimeter of callosal connectivity), which we deduce to have been into area V6A (S. Shipp, M. Blanton and S. Zeki, manuscript in preparation; and see below). The nature of the change in laminar organization is consistent with the general scheme that describes successive levels within a hierarchy of cortical areas (Felleman and Van Essen, 1991), and it implies that V6A is at least one step beyond V6 in the chain of cortical processing. Figure 31 shows a three-dimensional reconstruction of one example with an injection into the dorsal part of the caudal face of SPG, which resulted in a number of bands of connectivity aligned dorsoventrally over the superior parietal gyrus. The laminar organization at the points marked A and B is discernibly different; A shows a relatively even distribution of terminal label over all layers, whereas at B this distribution is more focused upon layers 1, 5 and 6, and is minimal in layer 4 (Figure 311). The latter is characteristic of a descending connection and has been described for many pairs of areas whose serial arrangement is not in doubt (Felleman and Van Essen 1991). The former, more evenly distributed pattern is typical of an intrinsic connection within an area, though it has been less frequently reported (e.g. Rockland and Pandya, 1979; Maunsell and Van Essen, 1983a; Rockland, 1985). Retrogradely filled cells occur in the supragranular and infragranular layers at both A and B, a pattern of labelling that is non-specific with regard to hierarchical relationships (Felleman and Van Essen 1991). We draw the border between V6 and V6A along the transition between pattern B and pattern A. It is not necessarily abrupt, but can normally be localized

6 Areas V6 and V6A in the SPG of macaque 35 to within a range of several hundred micrometres. The border was roughly parallel to the plane of section, and at a level that falls just below the ventralmost extent of the injection site, leading to the conclusion that the injection is wholly sited within the more dorsal area, V6A. Figure 4 shows a second example, a case in which such a transition could be recognized at two separate locations: one approximately corresponding to that in Figure 3, and a second on the medial side of the gyrus, at a more ventral location. The simplest assumption is that the V6N6A border runs obliquely over the gyrus between these two locations. If so, it also passes through the region of WGA-HRP deposition, implying that the ventral part of the injection site and its accompanying shell of intrinsic connections should be attributed to V6. However, this did not noticeably affect the precision with which we could locate the two sites of transition (from intrinsic to descending laminar characteristics) at the presumptive V6N6A border. A reconstruction of the labelling resulting from a third injection into V6A is shown in Figure 5. This did not reveal a transition as such, since the bands of label towards the foot of the gym were not continuous with the injection site. However, the labelling towards the foot of the SPG was uniformly of descending character, which we interpret to signify that it was entirely within area V6. Furthermore, this patchily distributed pattern of labelling had a well defined upper limit, which provides a conservative estimate for the upper border of V6 in this case; the border might be dorsal to this estimate, but not ventral. A comparable method of establishing the border between V6 and V6A is by examining the pattern of inputs from a third area, for instance V5. Figure 6 shows one such example, following an injection into a region of far peripheral representation within V5 [this is case SP 27 as documented in Shipp and Zeki (1989)l. Here it is V6 that contains a relatively diffuse laminar distribution of terminals, an intermediate pattern thought to distinguish a so-called lateral connection, between areas of equal rank (Maunsell and Van Essen, 1983a; Andersen et al., 1990a; Boussaoud et al., 1990). Ungerleider and Desimone (l986a) have also previously identified this projection (from MT to PO in their terminology) as being of intermediate character. Dorsal to this intermediate pattern, the terminals show a greater concentration in layer 4, indicative of an ascending connection from V5, presumably to V6A. The reconstruction in Figure 61 shows that the region of labelling was continuous across the intermediate and ascending zones and thus the point of transition (indicated by long arrows) provides an accurate localization of the prospective border between V6 and V6A. Ventral to V6, the connections adopt an evident descending character, possibly representing a connection with area V3 (as labelled in Fig. 611). And latero-dorsal to V6A. in a separate patch of labelling further within the intraparietal sulcus, the terminals were more or less restricted to layer 4, a very obvious ascending pattern of connection that might represent area MIP of Colby et al. (1988). These three-dimensional reconstructions were resliced (see Materials and methods) into the parasagittal plane, in order to construct two-dimensional maps of identical format to those used to summarize the physiological data (see below). The two-dimensional maps permit a ready comparison between different individuals-by direct superimposition-which is prohibited by substantial variations in the shape of the three-dimensional reconstructions. Figure 7 shows two-dimensional maps constructed from each anatomical case. The left part of Figure 8 provides a summary superimposition, showing that the four estimates of the border span a range of -2 mm within our normalized map of the SPG. Some variation is clearly to be expected but this level of consistency is as good as could be expected, and certainly allows the connectional demarcation of the border to be compared to the architectural and physiological versions. Possible architectural borders Other studies of this region of the cortex have emphasized myelination as a criterion for defining area PO (Covey et al., 1982; Gattass et al., 1985; Colby etal., 1988). As described below, PO was not especially obvious in our own histological material, but in seeking to confirm the criteria for PO we discovered other architectural features within the caudal SPG that were more prominent, and which we illustrate in Figure 9. The most obvious of these was a difference in the density of myelination of layer 1 between the posterior and anterior banks of the POS. The anterior bank (the region between the two black arrows in Fig. 9, top left) has fewer horizontal fibres in layer 1. This is true for both components of layer 1, the superficial dense fibre plexus and the fibre sparse region that separates it from layer 2. The decrement in fibre density in layer 1 is relatively sharp in the fundus of the POS (Fig. 9A), and its recovery towards the crown of the SPG is relatively more gradual (Fig. 9B). The fibre sparse region of layer 1 appears to demarcate a zone in the anterior bank of the POS that is comparable to cytoarchitectural area 19 of Brodmann (1905) and area OA of von Bonin and Bailey (1947). All these authors have referred to the cytoarchitectural borders as being diffusely located, which accords with our own observations in Nissl-stained sections; the frequency of large pyramidal cells in layer 5, for instance, certainly increases around the crown of the SPG away from the sulcus (i.e. from area OA to area PE), but at no point does this feature allow one to pinpoint an obvious border. The myelin sections are more satisfactory in this regard, and we found two other features that seemed to correlate with the increase in fibre density in layer 1, and that could distinguish area PE from OA: the density, and perhaps length, of tangential fibres, and the prominence of radial fibres. The former is responsible for the two bands of Baillarger appearing a little more dense in PE; we see nothing in these bands that might be described as faint (Pandya and Seltzer, 1982). Overall, PE has heavier myelination than OA. Provisionally, we suppose that the OAPE border is equivalent to the V6ME border. Are there any features which might indicate the border of V6 with V6A? We found, at roughly the expected location, a point where the radial fibres become less linear and parallel, and more clumped together and irregularly spaced, most obviously in layers 5 and 6 (Fig. 9D; the white arrowhead shows the point of transition to the more irregular pattern, at right, possibly V6A). The same change is reflected in Nissl sections, where the regular matrix of cells in layers 5 and 6 of V6 is distorted into more irregular columns, spaced by wider gaps, more dorsally. We plotted this location in a series of parasagittal sections to produce a two-dimensional reconstruction, again in the format of Figure 1. The result of this work is reported in the right part of Figure 8, also showing the architectural transitions corresponding to the two black arrows in Figure 9. The putative V6/ V6A border that arises from this process is within the range of our other determinations of the V6N6A border (compare left and right parts of Fig. 8, and see the summary Fig. 17D), although none of our primary experimental material allows us directly to equate the two. The architectural staining quality of the physiology brain tissue was impaired by the numerous electrode penetrations. And the anatomy (WGA-HRP-injected) brains were sliced in a horizontal plane that intersects the border very obliquely, so that it had to be detectable in just one or two sections, or else by an unreliable Comparison between sections.

7 36 Areas V6 and V6A in the SPG of macaque Colby et al. (1988) define PO by its heavier myelination, and remark on the presence of horizontal fibres. Neither feature was particularly evident in our material, certainly not for the purpose of tracing a border. One might note that there is a minimum in the density of myelination about halfway up the anterior bank, and a maximum -3 mm more ventral (roughly coincident with the upper and lower margins of frame D respectively); a density border might be placed anywhere between these two points, but its exact location would be arbitrary. More ventrally the density of myelination recedes into the fundus of the sulcus, one of the normal sequelae of cortical curvature as the superficial layers gain in relative thickness. The radial character of the fibres diminishes concurrently (indicated by the arrowhead in Fig. 9C). Also in this region there is a cytoarchitec- tural change in layer 2 (it becomes less condensed), seen in the Nissl stain. Finally, as described above, there is the resumption of relatively dense myelination of layer I, just beyond the point of maximum curvature in the fundus, towards the posterior bank. Whether this (or any of the other) architectural features serves to denote the lower border of V6 is uncertain, for we have no other, more substantive, criteria with which to assess it. Physiological location of the V6N6A border We have made extracellular recordings from the caudal aspect of the SPG in four hemispheres of three awake macaque monkeys, altogether studying 1152 cells. We first tested each unit with classic visual

8 Areas V6 and V6A in the SPG of macaque 37 FIG. 3. (I) A macaque left hemisphere dissected to afford a view of the superior parietal gym (SPG) comparable to that shown in the three-dimensional reconstruction reported below. The exposed wall of the gyms (highlight) is repictured below as an inset to show the division between the intraparietal and parieto-occipital sulci (IPS and POS). The SPG was injected with WGA-HRP (case SP 24L). The injection site is shown in broken outline in the threedimensional reconstruction; other details as in Figure 2. The arrows A and B refer to sites illustrated in 11. (11) Dark-field photomicrographs of labelling at positions A and B in I. The overall density of labelling is not dissimilar at these two locations. But note that in A terminal labelling is fairly uniform across layers-perhaps heaviest in layer 5, lightest in layer 2 and about equally dense in layers 1, 3, 4 and 6; this is an intrinsic connection. In B, by contrast, terminal label is relatively more dense in layer I, and relatively less dense in layer 4; this is a descending projection. FIG. 4. Three-dimensional reconstruction of an entire hemisphere dissected as in Figure 31 to reveal the distribution of transported label on the anterior banks of the parieto-occipital and intraparietal sulci (POS and IPS). The site of injection of WGA-HRP (shown in broken outline) involves both V6A and V6 (case SP 25L). Connections with the frontal premotoi cortex are also visible, around the arcuate sulcus (AS). (Inset) Medial view (anterior is on the right) of the same injection, showing connections to the medial wall of the hemisphere between the cingulate and medial parieto-occipital sulci (CS and mpos). Arrows point to mansitions between the intermediate and descending laminar patterns of labelling. The lower transition point is visible from both angles of view. Other details as in Figures 2 and 3. IOS, inferior-occipital sulcus; STS, superior temporal sulcus.

9 38 Areas V6 and V6A in the SPG of macaque FIG. 5. Three-dimensional reconstruction of the posterior part of a right hemisphere 'dissected' as in Figure 31 to reveal the distribution of transported label on the anterior banks of the parieto-occipital and intraparietal sulci (POS and IPS). The site of injection of WGA-HRP (shown in broken outline) was probably restricted to V6A (case SP 14R). (Left) Postero-medial view (anterior is to the left) of the brain. (Right) Postero-lateral view (anterior is on the right) of the brain. The two viewpoints show the widespread distribution of label towards the foot of the SPG, also extending onto the medial surface; the laminar characteristics of this label were uniformly typical of a lower area, and it earmarks the likely territory of area V6 in this case. Also visible are dorsal and ventral patches of label in the anterior (medial) bank of the IPS. Other details as in Figures 2 and 3. mpos, medial parieto-occipital sulcus.

10 Areas V6 and V6A in the SPG of macaque 39 visual background over part of the screen, or even just switching on and off the screen background illumination. In spite of our efforts, visual responses were elicited in only 66% of SPG cells (765/1152), the remainder (387/1152) being completely insensitive to any kind of visual stimulation that we used. We decided to classify these latter cells as non-visual neurons. It is possible that they may have been responsive to stimuli that were not part of our repertoire, but nevertheless they can be considered a distinct functional class, by default. Two-dimensional maps of the recording sites in the SPG showed that the non-visual neurons were restricted to the dorsal part of the caudal SPG, whereas the visual neurons were more widespread. Figure 10 shows two-dimensional maps of the distribution of visual/ non-visual neurons in the individual hemispheres and, at right, a superimposition of data from all four. The summary map clearly shows the non-visual units to respect a ventral boundary that is angled somewhat obliquely (from dorso-lateral to ventro-medial) across the posterior face of the SPG. This same feature is also very obvious for the individual case (1 5L) in which the greatest number of recordings was obtained. It seems reasonable to suppose that the lower limit of the non-visual cells marks the location of the border between two separate functional areas, notionally areas V6 and V6A. To be more precise, it marks the uppermost location of any such border, since the distribution of non-visual cells is somewhat sporadic, and there is no guarantee that the last non-visual cell recorded in any penetration passing from dorsal to ventral was precisely coincident with the putative functional border. From the above we conclude that the property of visual unresponsiveness (or, to be conservative, cryptic visual selectivity) is a characteristic of the dorsal area V6A. Careful examination of the cortical distribution of other cell characteristics revealed various functional properties which share a similar distribution. The latter include certain kinds of complex visual selectivities and various extraretinal factors that were also found to govern the response level. Other more basic visual properties (such as orientation and direction selectivity) that are familiar in earlier visual areas were not segregated. We describe these properties and their distributions in turn. FIG. 6. (I) Top left three-dimensional reconstruction of a left hemisphere (case SP 27L) dissected as in Figure 31 to reveal the distribution of label over the SPG following an injection of WGA-HRP into area V5 (hidden within the fundus of the superior temporal sulcus; not indicated). The boxed area is enlarged to show, separately, the distribution of axonal terminal label in layers 1, 4 and 6. The long arrows indicate the level of a transition in the laminar patterning of this label that we interpret as the border between V6 and V6A. Short arrows point to sites illustrated in It. Other details as in Figures 2 and 3. (11) Photomicrographs to show the laminar pattern of labelling at sites we interpret as V6 and V3, indicated by the upper and lower thick arrows respectively in I. Terminal label is fairly evenly distributed across the cortical thickness in V6-this is an intermediate pattern. In V3 there is a massive concentration of terminal label in layer 1, and a relative avoidance of layer 4; this is a descending pattern. stimuli, such as spots or bars of light of variable size, orientation and direction of movement, in order to locate and plot a visual receptive field. If these stimuli were ineffective we resorted to a variety of other procedures, using simple or complex black figures, a textured Basic visual properties The sensitivity of visual cells to orientation, direction and speed of stimulus movement was tested by light or dark bars, or by single lightldark borders, moved across the receptive field. We defined a cell as orientation-selective if it failed to respond when the stimulus orientation differed by no more than 40 from the preferred orientation, orientation-sensitive if it ceased responding at stimulus orientations differing by from the preferred orientation, and as nonoriented if it continued to respond to all stimulus orientations. As far as direction sensitivity is concerned, we defined as directionselective those cells whose response to a correctly oriented stimulus moving in the direction opposite to the preferred one was <20% of the firing rate evoked during optimal stimulation; direction-sensitive those whose response was between 20 and 80%; and non directionsensitive those whose response in the opposite direction was >80% of that in the preferred one. Classified in this way, the great majority of visual cells of the caudal part of SPG was either orientation-selective or directionselective, or both (Fig. 11). However there was no sign of any spatial segregation of these properties in the caudal SPG: selective, sensitive and non-sensitive classes were found in roughly equal proportions either side of the non-visual boundary line.

11 40 Areas V6 and V6A in the SPG of macaque SP 24L SP 25L SP 14R SP 27L FIG. 7. Two-dimensional reconstructions of the V6N6A border (thick dashes) derived from the four brains illustrated in Figures 3-6 (from left to right respectively). Two-dimensional maps of individual cases were reconstructed from the parasagittal sections obtained by reslicing the three-dimensional reconstructions of single cases, as described in Figure 2. The grey area corresponds to the cortex of the anterior bank of POS; all other details as in Figure 1. Cells were also classified according to their tuning for stimulus speed, and we found one class, slow cells, that was clearly segregated in the dorsal aspect of the SPG. Slow (S) cells were classified as cells optimally responsive to static visual stimuli and to stimuli moving at very slow speeds; they were unresponsive to speeds of 310 /s. Slow cells were distinguished from slow-medium cells, which became unresponsive at speeds between 10 and 100 /s, and from slow-medium-high cells, which continued to respond to speeds > 100 /s. Other classes were medium-high cells, which responded to any stimulus moving at >loo/s, and high (H) cells, responding only to speeds greater than 100 /s. Figure 12 shows the relative frequency of these classes, the least speed-sensitive class, SMH, being the most common. Their distribution over the caudal SPG is illustrated by Figure 18A, which shows that only the two extreme classes, S and H, were spatially segregated, both being restricted to the dorsal zone. Since there were only seven cells classed as H, we regard the slow cell class (S) alone as a potential marker for V6A. connectional border architectural borders Complex visual properties Some visual neurons of the caudal SPG ( ) were unresponsive to the simplest visual stimuli, such as lighvdark borders, light/dark bars, and spots, and required more specific forms of stimulation. Figure 13 shows a typical example. This cell did not respond to a vertical lighudark border horizontally moved to and fro across the receptive field but it gave reliable, brisk responses to the same movements of a light/dark comer. Using the comer stimulus, the boundary of the receptive field could be plotted quite accurately, although the position of the comer within the receptive field was not critical. This cell was in our complex visual responses category. Other examples were activated by static, single or double comers at a certain optimal orientation; in these cases strong tonic responses were maintained for the duration of visual stimulation. Finally, for the most intransigent visual cells we were not able to find a specific configuration of a comer, or comers, able to activate the cell reliably, but we did observe that complex shadows continuously changing form and direction of movement could elicit a significant response. To be effective, these complex visual stimulations had to be performed FIG. 8. Connectional and architectural borders in the caudal face of the superior parietal gyrus. (Left) Summary two-dimensional reconstruction showing a superimposition of all four locations of the V6N6A border as determined by analysis of the laminar patterns of cortico-cortical connections. Crosses (+) correspond to the border of case SP 24L, thick dashes to SP 25L, triangles to SP 14R and X to SP 27L, all of them shown in Figure 7. Three cases were rescaled to match the dorso-ventral extent of the SPG in case SP 14R, which provides the basic contour map, Since most individual cases were represented by left hemispheres, the basic contour map was reflected to match the left hemisphere format. (Right) Two-dimensional reconstruction of a single case showing three architectural borders. A (triangles), B (-) and D (+) correspond to the borders illustrated in the frames enlarged in Figure 9. The relevant architectural features of each are described in the text. Border D approximates to V6N6A as determined by connections, at left. The grey area corresponds to the cortex of the anterior bank of POS; all other details as in Figure 1. within a definite region of space, allowing us to plot an approximate visual receptive field for these cells. Another set of cells, which we have grouped with those described

12 Areas V6 and V6A in the SPG of macaque 41 FIG. 9. A parasagittal section through the parieto-occipital sulcus with selective enlargements to illustrate architectural changes around the posterior rim of the superior parietal gyms. On the top left, the parieto-occipital sulcus is shown (right = anterior; up = dorsal) with frames indicating the cortical areas enlarged in A, B, C and D. (A) Arrow indicates a sharp transition in the density of fibres in layer 1 within the fundus of the POS; the relatively light myelination of layer I is a characteristic of most of the anterior bank of the POS. (B) Arrow indicates the approximate location of the prospective border of V6A with area PE. There is an increase in the density of fibres in layer 1 from this point towards the right, and also a notably greater number of long horizontal fibres, in all layers. (C) Light arrow indicates a point at which long radial fibres become prominent. The same point in an adjacent section stained for Nissl substance is shown above. Also visible in the Nissl section is a modest cytoarchitectural change, in that layer 2 becomes more condensed at more dorsal levels. (D) Arrow indicates a transition from long regular radial fibres (on the left) to shorter more clumped fibres. The same transition can be recognized in the adjacent section stained for Nissl substance (above), where the serried columns of cells in layers 5 and 6 become a little more irregular, Scale bars: top left, 2 mm; B, 500 p n (applies to A-D). above, was not unresponsive to simple visual stimuli, but they nevertheless showed a complex visual behaviour. Figure 14 illustrates a neuron that failed to respond to a light or dark bar moved across the receptive field (Fig. 14A) but was readily driven by a lighudark border, with strong direction selectivity (Fig. 14B). When we reversed the lighddark border the neuron accordingly reversed its preferred direction (Fig. 14C). As a logical consequence of this behaviour, expansion of a dark bar centred in the receptive field was very effective in activating this neuron (Fig. 14D), but the opposite contraction was not (Fig. 14E). Figure 15 shows the cortical distribution of all SPG neurons with complex visual properties. It is quite clear that these neurons were concentrated towards the dorsal end of the recording zone on the caudal SPG, none of them being located in the ventral part of it. Extraretinal modulation In many cells of the caudal SPG the act of fixating during a fixation task changed the discharge rate of the cell. Generally the discharge rate decreased after fixation, as shown in Figure 16. The change in discharge rate after fixation was not related to particular directions of gaze, although many neurons of this cortical region were affected by gaze direction (Galletti et al., 1995). It seemed related to sustained fixation of the fixation target and not to fixation per se, since changes in the discharge rate of these cells were not evident during free visual searching of the animal in darkness. In other words, the activity of these cells may reflect the monkey s level of attention to its operant task, which is presumably much diminished in the intervals between routine visual testing, and equally likely to differ during periods of free and active fixation. Often the fixation effect appeared to adapt

13 42 Areas V6 and V6A in the SPG of macaque case 13 L case 14L Summary of four cases case 15L case 15R o visual cell 0 non-visual cell FIG. 10. Two-dimensional maps from each individual and (right) a summary map of all four to show the spatial segregation of visual (n = 765) and non-visual (n = 387) units on the caudal SPG. For the summary superimposition, case 14L was rescaled and case 15R reflected. The summary uses the individual contour map from case 15L, which provided the greatest volume of data. The grey areas correspond to the cortex of the anterior bank of POS; all other details as in Figure 1. (i.e. the discharge rate regained its spontaneous level) over the course of a series of trials in which the monkey s task remained constant; in such cases the fixation effect was generally restored if the monkey s task was altered, or after a break in the sequence of testing. Observations of this nature are also consistent with the hypothesis that the fixation effect is related to fluctuations in attention. In Figure 17 we show the responses of a neuron which demonstrated the fixation effect particularly clearly, but only in circumstances where we deliberately manipulated the monkey s level of attention. The spontaneous discharge rate of this neuron was quite high both in darkness and light, even during the fixation task (Fig. 17A, B). Visual stimulation near the fovea delivered during fixation produced transient inhibition, the cell s rate of discharge returning to normal within a few trials (Fig. 17C). A sudden change in the direction of stimulus movement (arrows in Fig. 17C) briefly restored the inhibition, but the discharge rate again rapidly recovered. Only by continuously changing the form and direction of visual stimulation (using hand movements along the optical line behind the screen) was it possible to maintain consistent inhibition of the cell s discharge (Fig. 17D). It was very clear during this manoeuvre that if we persisted too long with any one form of visual stimulation, the cell was likely to recover its normal high rate of discharge. The behaviour shown in Figure 17C and D seems to imply that the cell s discharge rate is maximally inhibited when the visual stimulus is most novel, and hence distracting. This would actually be consistent with the fixation effect, as described above, since this is when the monkey would have to expend the most effort in maintaining fixation upon the fixation target, in order to perform its task correctly. The tests shown in Figure 17E were an attempt to demonstrate this more directly, by manipulating the monkey s attention without the use of novel visual stimuli. To do this we induced an error in the monkey s performance, simply by extinguishing the fixation spot in mid-trial. Since in several cases we switched off the fixation target for a while during the trials (Galletti et al., 1995), the monkey was used to this event and maintained its direction of gaze after target extinction waiting for target-on. But this time the spot remained invisible, so the animal was unable to detect the change in colour of the spot (its cue to press a lever for reward). The error was

14 Areas V6 and V6A in the SPG of macaque NOR OSN OSL 0 X 0 -. NDS DSN 0 X DSL 0 I I I I I FIG. 11. Frequency and spatial distribution over the caudal SFG of orientation tuning (A) and direction tuning (B). The data derive from all four cases studied. NOR, non-oriented cells (n = 27); OSN, orientation-sensitive cells (n = 88); OSL, Orientation-selective cells (n = 144); NDS, non-direction-sensitive cells (n = 63); DSN, direction-sensitive cells (n = 9); DSL, direction-selective cells (n = 162). All these cells are plotted in the two-dimensional maps shown in the bottom. The grey areas in two-dimensional maps correspond to the cortex of the anterior bank of POS; all other details as in Figure 1. indicated by a sound-the usual signal for a mistake-with the expectation that the monkey should perform the subsequent trial, conducted in routine fashion, with a raised level of vigilance. Two such post-error trials are indicated by arrows in Figure 17E, and it can be seen that the cell s discharge rate was indeed strongly inhibited in these trials, as we predicted. In all the trials following induced mistakes we observed 100% correct performance and a shorter reaction time, observations which tend to confirm that the monkey s attentional state was enhanced during these trials. Figure 15 shows the cortical distribution in the caudal SPG of the neurons that showed signs of modulation by the level of attention. They were clearly segregated in the dorsal aspect of caudal SPG (V6A). None of them was located in the ventral part of it. In previous papers (Galletti et af., 1993, 1995) it has been reported that the activity of many neurons of the caudal portion of SPG is modulated by the direction of gaze, and that a number of them have visual receptive fields that remained anchored to the same spatial locations regardless of gaze direction (real-position cells). Figure 1 8B shows that gaze-sensitive neurons are evenly distributed across the caudal SPG while real-position cells are segregated in the dorsal part of it. Figure 18C combines all the visual and extraretinal functional properties which appear to cosegregate in the dorsal part of the caudal SPG. The distribution of these units respects the same ventral limit as the property of non-visual responsivity, and their addition to the visualhon-visual map clarifies the resolution of this borderline, our

15 44 Areas V6 and V6A in the SPG of macaque physiological determination of the V6N6A border. In Figure 18D we have drawn together all three approaches for locating the border: physiological, connectional and architectural. The outcome is that the net physiological border falls within the range determined anatomically. Thus the discrepancy between these techniques is no greater than the range of variation obtained by applying the same technique to different individuals. We conclude that, within the limits of cartographic resolution, the anatomical and physiological borders are entirely consistent. Given that the three criteria are totally independent, and originate in separate laboratories from nine different hemispheres, unit 15, S SM SMH MH H (degreedsec) -,,,., I. 400 msec/division FIG. 12. The relative frequency of cells tuned to different speeds (pooled data from all four cases). S, cells sensitive to slow speeds (O-lOD/s); SM. cells sensitive to slow to medium speeds (0-100"/s); SMH, cells sensitive to slow, medium and high speeds (0-900"ls); MH, cells sensitive to medium to high speeds (1O-90O0/s); H, cells sensitive to high speeds (1W90Oo/s). FIG. 13. Responses of a cell recorded in the dorsal part of the caudal SPG to edges and comers horizontally moving 'to and fro' across the receptive field (dashed circle). Crosses indicate the animal's fixation point and arrows the directions of stimulus movement. Cell's responses are reported as peristimulus time histograms. unit 15,078 FIG. 14. Responses of a cell recorded in the dorsal part of the caudal SPG to moving bars (A), edges (B and C), and the expansion (D) or contraction (E) of a dark bar. Conventions as Figure 13. Scales are eight spikes per vertical division and 400 ms per horizontal division.

16 Areas V6 and V6A in the SPG of macaque 45 we are confident that, at least within the region of the gyrus where our studies overlap, the border of V6 and V6A is sufficiently established and reliable. Statistical comparison of receptive field size and scatter in V6 and V6A For the purpose of this analysis all the cells that we studied were classified as 'ventral' or 'dorsal' (V6 or V6A), reflecting the map of segregated functional properties in Figure 18C. Cells recorded within penetrations whose total excursion was clearly restricted to either the ventral or dorsal sector (as determined from the two-dimensional maps of individual cases) were classified accordingly. Penetrations which passed through both sectors were subdivided according to the principle that any cell showing at least one of the 'dorsal' properties from Figure 18C was allocated to the dorsal group (V6A), along with all the preceding units in that penetration. Thus the ventral group (V6) was a partially default classification, consisting of units recorded below the level of the final dorsal unit in any given penetration, in o simple visual 0 complex visual x attention I FIG. 15. Two-dimensional map showing the distribution over the caudal SPG of cells activated by simple visual stimuli, cells with relatively complex visual selectivities, and those whose discharge showed signs of attentional modulation. Data pooled from all four cases studied. The grey area corresponds to the cortex of the anterior bank of POS; all other details as in Figure 1. uut msecldivision FIG. 16. Changes of a cell's activity during the animal's fixation ('fixation effect'). Neuronal activity was collected in complete darkness while the animal fixated a small spot of light for several seconds. The cell's activity is shown as a peristimulus time histogram and sequences of action potentials. In the bottom part of the figure, x and y components of eye position during all trials are also reported. Y u I.,,.,,,,,, 1 unit msec/division FIG. 17. Activity of a cell recorded in the dorsal part of the caudal SPG that was specifically tested to investigate the relationship of cell discharge to the level of attention. Crosses and squares on the left indicate the fixation point and screen in front of the animal, respectively. Rasters on the right indicate the cell's activity (sequences of action potentials). (A and B) Spontaneous activity during fixation, (A) in the dark and (B) with uniform background i~lumination of 2 cd/m2. (c) cell's activity to repetitive axial motion of a dark rectangle. Arrows show the activity during trials immediately following a change in the axis of stimulus motion, i.e. from horizontal to oblique and from oblique to vertical. (D) Cell's activity to irregular, continually varied hand movements. (E) Spontaneous activity during fixation in light; arrows indicate the activity in trials that followed immediately after a monkey error.

17 46 Areas V6 and V6A in the SPG of macaque

18 Areas V6 and V6A in the SPG of macaque 47 addition to those recorded within wholly ventral penetrations. Mean receptive field sizes and scatters were then calculated separately for the dorsal and ventral classes of units within each penetration. Figure 19 shows the resultant plot of mean receptive field size, and scatter, against mean receptive field eccentricity for the two groups of data. Values of receptive field size in the dorsal group (likely V6A) were on average -10" larger than in the ventral group (likely V6) at any given eccentricity. The associated level of significance was P < The difference shows negligible dependence on eccentricity, as the test for a difference in slope (or regression coefficient) of the two data sets was negative, with P > 0.1; hence Figure 19 plots a model of regression in which the slopes are constrained to be identical. A difference in receptive field size of 10" is equivalent to the mean receptive field size in V6A, being -50% larger than that in V6, at the median eccentricity of this data set (25"). It is notable, however, that there is substantial overlap between receptive field sizes in the two areas; a measurement of an individual cell's receptive field size is, by itself, incapable of distinguishing the location of the recording site. Thus our data show receptive field size to be a comparatively poor factor for localizing the border between V6 and V6A. The comparison of receptive field scatter between the two data sets gave a similar outcome, illustrated in the lower half of Figure 19. Receptive field scatter was greater for the dorsal receptive fields by -5" at any given eccentricity, and the result was significant at P < There was no difference between the slopes (P > 0.1). It is also notable that both receptive field size and receptive field scatter increase with eccentricity, a finding of very general applicability in areas of visual cortex, but not of areas PO and Pod, as reported by Neuenschwander et al. (1994) for the corresponding ventral part of the caudal SPG in the Cebus monkey. Discussion Areas of the cerebral cortex are conceived as discrete zones with distinct functional characters; dissimilarities in function between visual areas are reflected by their different visuotopic organizations, architectures, connectivities and, most directly, by their populations of neuronal response properties. A cornerstone of this concept is that all these criteria should give coincident boundaries for an area. If not, the criterion which most meaningfully defines a functional zone should take precedence-in which case direct recording of neural response properties must be the final arbiter of any territorial dispute. But the veracity of such physiological data is usually matched by its scarcity: hence, in order of convenience, the techniques of simple physiology (receptive field mapping), connectional studies and histology (cortical architecture) are more commonly substituted for areal demarcation. It remains a drawback that these techniques may reveal little, if anything, about the nature of the functional change at the borders they reveal. Connectional studies are arguably the most potent in this respect, as the functional characteristics of regions shown to be anatomically linked may already be known, and the relative n Ln 60.0j % v I I 0 V6A 9-b / + / t 10.0 "..,. "..,, mean eccentricity (degrees) FIG. 19. Receptive field size and scatter versus eccentricity in V6 and V6A. (Above) Dual regression plot of receptive field size against eccentricity for groups of units recorded in V6 or V6A (16 and 26 penetrations respectively). Each point represents the mean receptive field size and eccentricity for all units recorded in one penetration; samples with less than four units have been excluded. The plotted data represent 120 separate receptive fields recorded from V6 (an average of 7.5 units per penetration) and 189 receptive fields recorded from V6A (an average of 7.3 units per penetration). Using a 'pooled slope' analysis of covariance model which constrains the regression coefficients to be identical, the regression equations are: V6: size = 0.33ecc '; V6A: size = 0.33ecc ". The mean difference in redeptive field size between V6 and V6A is computed to be 10.3". for which F1.39 = 9.9 (P = ). (Below) Dual regression plot of receptive field scatter against eccentricity for the same groups of units we used in receptive field size analysis. Using a 'pooled slope' analysis of covariance model which constrains the regression coefficients to be identical, the regression equations are: V6: scatter = O.15ecc + 2.1'; V6A: scatter = O.15ecc + 6.7". The mean difference in receptive field scatter between V6 and V6A is computed to be 4.6", for which F1.39 = 6.3 (P = 0.016). hierarchical standing-inferred from an analysis of the laminar pattern of connections-may help in understanding the functional role of the connected structure. The aim of the current exercise has been to 0 0 FIG. 18. Functional maps in the caudal pole of the superior parietal gyrus. (A) Two-dimensional map showing the distribution over the caudal SPG of five different classes of speed tuning; classes as in Figure 12. (B) Two-dimensional map showing the distribution of cells influenced by direction of gaze, and of 'real-position' cells. (C) Two-dimensional map showing the distribution of all functional properties which are codistributed with the non-visual cells and which appear to be characteristic of area V6A. (D) Final summary map showing physiological, connectional and architectural estimates of the border between V6 and V6A. Data in A, B and C derive from four hemispheres, data in D from nine hemispheres. The grey areas correspond to the cortex of the anterior bank of POS; all other details as in Figure 1.

19 48 Areas V6 and V6A in the SPG of macaque combine connectional studies and recording of neural response properties in the definition of a truly functionally characterized border. As it happens, the region of cortex we have studied, the posterior aspect of the SPG, is a region previously characterized by myelin staining and receptive field mapping in the demarcation of area PO (Covey er al., 1982; Gattass et al., 1985; Colby et al., 1988). We have examined both factors and found both to be relatively poor tools for locating the exact border of PO. Instead, we have attempted to marry an anatomical analysis of the local connectivity of this same region (S. Shipp, M. Blanton and S. Zeki, manuscript in preparation) with an extensive set of physiological data gathered from repeated recordings in alert monkeys (Galletti et al., 1991a, 1993, 1995). We have found two separate areas in this cortical region, named V6 and V6A. Although collating data from a number of separate hemispheres poses its own problems, the putative physiological and anatomical boundaries between these two areas were found to coincide, at least within the range of individual variation and the topological resolution of our reconstruction techniques. Connectional determination of the V6N6A border The anatomical determination of the V6N6A border depends upon a close analysis of the laminar pattern of labelled axonal terminals following injections of WGA-HRP. Ascending and descending connections have different laminar distributions, the former concentrating in layer 4, and the latter avoiding layer 4 to concentrate in layers 6, 5 and 1; intrinsic connections have a pattern intermediate between these two (Rockland and Pandya, 1979, Maunsell and Van Essen, 1983a; Felleman and Van Essen, 1991; S. Shipp, M. Blanton and S. Zeki, manuscript in preparation). Thus, if a patch of transported label possesses a clear ascending or descending laminar character, it is evidently in a different area to the injection site. We used this simple principle to identify segments of the V6N6A border in two ways. Firstly, injections into the SPG itself (centred within V6A) produced bands of label stretching ventrally away from the site of injection, and the zone of transition where the laminar pattern changed from intermediate to descending character indicated the border with V6. Secondly, we examined a case with an injection into peripheral V5 that had produced an equally broad distribution of labelling over the posterior aspect of the SPG. This possessed an ascending character dorsally, becoming intermediate, and eventually descending in laminar character toward the foot of the gyrus. In this case the V6N6A border is likely to be represented by the transition from intermediate to ascending laminar character, since areas of equivalent hierarchical status are known to communicate with each other by intermediate laminar patterns of connection (Maunsell and Van Essen, 1983a); the likelihood is that V6 is represented by the intermediate region, that V6 and V5 occupy the same level in the cerebral hierarchy of areas, and that V6A, one rank up in the hierarchy, is represented by part of the region of ascending character. Physiological determination of the V6N6A border The chief physiological criterion for demarcating the dorsal region was the presence of units that were unresponsive to any form of visual stimulation, as previously reported for a smaller quantity of data (Galletti et al., 1991a). We refer to these cells as non-visual, though the term is clearly provisional in that some form of visual stimulation currently absent from our experimental repertoire could conceivably be sufficient to activate these cells. Such a discovery would not invalidate our use of non-visual cells to delimit a border, since it is the asymmetrical distribution of these units that is the key feature; the physiological evidence would transmute to yield a distinction of purely visual character, but the location of the border would remain unchanged. The non-visual units were the most effective at describing a border line because they were the most numerous. But several other functional indices were also distributed asymmetrically about this line; these not only assist the demarcation of two separate areas, they also suggest that evolution of visual character does indeed take place between V6 and V6A. The nature of this functional distinction is one that we consider in more detail below. Relationship to the definition of area PO Area PO was initially described as a visual region on the anterior bank of the POS that could be recognized in a fibre stain (Covey et al., 1982). It was at first shown to occupy the full dorso-ventral extent of this bank (Gattass et al., 1985), but at a later date the more dorsal sector was excluded, due to its possession of larger visual receptive fields that were also less responsive to visual stimulation than those found more ventrally (Colby et al., 1988). Our data concur with this last observation, since we found V6A to possess larger receptive fields than V6. Yet PO in the macaque is currently defined primarily by its myeloarchitecture. It possesses clear inner and outer bands of Baillarger, though these do not readily distinguish it from neighbouring areas. The more distinctive features are said to be the density of myelination, which differentiates it from the dorsal area, though not from V3 nor V2, and the presence of numerous thick horizontal fibres in the lower layers (Colby et al., 1988). The relationship of receptive field size to density of myelination at the dorsal border of PO has yet to be documented in detail. We have sought to identify area PO in purpose-stained (Gallyas 1979) myelin material, with mixed results. For instance we saw no particular increase in the prevalence of horizontal fibres in the region of V6/ PO; this feature was only evident in area PE, several millimetres more dorsal at the top of the gyrus. Nor was area PO distinguishable by its density of myelination. The latter is a poor feature to use to identify cerebral areas for a number of reasons. Firstly, the density of silver impregnation can vary between sections, and even within the same section, due to technical deficiencies in the staining procedure. Secondly, cortical myelination varies with cortical gyrification and curvature, factors that must always be discounted. Finally, myelination density may vary within a unitary functionally defined area, the peripheral zone of reduced density, MTp, within area V5 providing one documented example (Ungerleider and Desimone, 1986b). We did, however, identify a different architectural feature, concerning the radial characteristics of myelination, that was roughly coincident with our other anatomical and physiological estimates of the V6N6A border. Several additional architectural features were also plotted, whose significance, if any, is unknown. Given that the relationship between specific architectural features and specific functional properties remains elusive, all these architectural boundaries are of unproven value until they are demonstrated to directly coincide with borders set by functional criteria. Difficulties of this nature may account for the fact that area PO, as defined by myelination, has repeatedly contracted in size. More recently, an area lying immediately dorsal to PO (Pod) has been described in the SPG of the New World Cebus monkey (Neuenschwander et al., 1994). Again the criteria for the definition are myeloarchitecture and receptive field mapping, and primarily the former: the report states that area PO is more densely myelinated

20 Areas V6 and V6A in the SPG of macaque 49 than area Pod, but that the border between them was not marked by any reversal or discontinuity of the receptive field mapping, nor any significant difference in receptive field size. This leaves only the far looser retinotopic criterion that each area should have a mostly complete representation of the contralateral hemifield. PO and Pod together are said to correspond to the PO complex of Gattass et al. (1985), and so the implication is one of a strict homology between area PO of Cebus and of Macaca (as defined by Colby et al., 1988), and of the existence of Pod in Macaca, although it has not been formally described in this genus. Nonetheless there are a number of discrepancies between the descriptions of area PO in the two genera. PO in Cebus has confluent bands of Baillarger, whereas in Macaca they are clearly separate. PO in Cebus is thus relatively densely myelinated, and distinct from Pod dorsally and V3 laterally. There is also a difference in location: the dorsal border of Pod in Cebus corresponds approximately to the location of the dorsal border of PO in Macaca [compare fig. 4 of Neuenschwander et al. (1994) with fig. 2 of Colby et al. (1988)l. Correspondingly, PO in Cebus is contained within the very foot of the SPG, and the area above Pod-identified as PEc of Pandya and Seltzer (1982)-is found some way down the gyrus, whereas in Macaca it is located much closer to the crown. We were thus led to consider the possibility that the regions defined as PO in these two genera do not directly correspond to each other. Evidence for this view derives not only from the relative locations of the two areas, but also from their physiology. Neuenschwander et al. (1994) found no significant difference in receptive field size between PO and Pod. This is unlike the report of Colby et al. (1988). where fields dorsal to PO are said to be larger than those in PO itself. Also Colby et al. (1988) report that cells dorsal to PO were very difficult to drive. Neuenschwander et al. (1994) do not mention recording visual fields dorsal to Pod, but they make no mention of fields in Pod being more difficult to activate than those of PO. Thus it seems possible that PO and Pod together in Cebus form the more accurate homologue of macaque PO. Certainly our own data show a clear overall increase in receptive field size in V6A relative to V6, and we also found V6A to present greater problems in finding a suitable stimulus for each cell. It is therefore unattractive to suppose that V6 and V6A in Macaca, as we have defined them here, correspond to PO and Pod in Cebus. Rather, it is more likely that PO plus Pod in Cebus correspond to V6 (PO) in Macaca. In a general sense it is not especially clear how plastic the interpretation of homology should be, in the face of the 30 million years or so of separate evolution that separate Cebus and Macaca from their common ancestor (Fleagle, 1988). The basic gyral morphology of the two genera is comparable, as are most of the basic features of the organization of visual cortex that have so far been described. One documented difference concerns a connection between V1 and lower V3 (V3v) in Cebus (Sousa et al, 1991), which is absent in Macaca (Van Essen et al., 1986), yet this would not be taken to imply that lower V3 is not a homologous field. Our point is rather that even when identical criteria are used, the correspondence of PO and Pod in Cebus to PO in the macaque is not unambiguous, and it would plainly be a disservice to the literature to employ these terms to describe our own definitions of cortical areas, derived from different criteria. This is the reason why a separate terminology has been retained. Is V6A a higher area than V6? The pattern of ascending and descending connections between V6 and V6A points rather directly to the fact that V6A should be considered the higher area-certainly in the purely anatomical sense of this term (Felleman and Van Essen 1991). Physiological correlates of elevated hierarchical status are somewhat diverse, but the evidence presented here is equally in accord with this notion. At a very basic level V6A has the larger receptive fields, a standard consequence of receiving topographically convergent ascending input (Zeki and Shipp, 1988). Selectivity for orientation and direction of movement may not be enhanced, but tolerance for higher speeds is one factor that may accompany larger receptive fields along a serial pathway (e.g. from V1 to V5 to VIP Maunsell and Van Essen, 1983b; Orban et al., 1986; Colby et al., 1993). Our data show that direction and orientation selectivity are comparable in V6 and V6A, and our classification of speed properties does not readily reveal a bias toward faster speeds in V6A. Paradoxically, in fact, we found V6A to possess an additional population of slow cells, unresponsive to speeds above 10 /s, that were absent from V6. The majority of slow cells (22/27 tested) were also direction-selective. Because the functional significance of these cells is uncertain, their presence in V6A is neutral with respect to higher function. A rather more telling index is the development of elaborate stimulus response selectivities, for example the presence of increasing numbers of real motion cells in V1, V2 and V3 (Galletti et al., 1984, 1988, 1990), or the evolution of pattern motion selectivity in V5 (Movshon et al., 1985; Rodman and Albright, 1989) followed, in MST, by selectivity for rotation and expansion (Saito et al., 1986; Sakata et al., 1986; Tanaka and Saito, 1989; Duffy and Wurtz, 1991; Orban et al., 1992; Graziano et al., 1994a). We found cells in V6A, but not V6, that were selective for one-dimensional expansion, and it is quite conceivable that the appropriate stimuli would have revealed properties not dissimilar to those of MST, given that the two areas are heavily interconnected (Colby et al., 1988; S. Shipp, M. Blanton and S. Zeki, manuscript in preparation). As it happens, we classified many other V6A neurons under the category complex visual responses that were driven (probably suboptimally in many cases) by irregular twodimensional deformation and translation, without always being able to specify the key stimulus features. Cells in V6A could also be more stringent with regard to the form of a stimulus; some, for instance, resembled the hypercomplex cells of Hubel and Wiesel (1965, 1968) in their selectivity for single- or double-cornered stimuli, but no such cells were encountered in V6. Another characteristic of higher visual function is the increased influence over neural activity of extraretinal factors, and again we found these to be rather more prominent in V6A than V6. The most awkward to encapsulate experimentally are those concerned with attention. In our experiments monkeys were trained to attend only to the fixation spot, and to ignore all other stimuli. Therefore we did not record any examples of a typical enhancement effect -where a non-foveated stimulus that becomes the subject of attention commands a much higher level of activity (Wurtz and Mohler, 1976; Bushnell et al., 1981). Rather, we found neurons in V6A whose activity seemed to correlate with the monkey s attention to its fixation spot. Ideally this attentional state should be constant, but naturally it will fluctuate as the monkey s vigilance waxes and wanes during the course of a recording session. Furthermore it is likely to fluctuate from trial to trial, depending how distracting a concurrently presented visual stimulus is, and thus how much effort (in some rather ill-defined sense) has to be expended in maintaining fixation in order to perform the operant task correctly. The behaviour of the cell shown in Figure 17 was best explained by this kind of attentional hypothesis: its firing rate was maximally inhibited in circumstances when a human observer

21 50 Areas V6 and V6A in the SPG of macaque performing the same task as the monkey would be expected to concentrate most heavily on the fixation spot. For instance, the cell s discharge rate decreased when confronted with novel visual stimuli, the effect diminishing as the novel stimulation was prolonged. The most natural inference is that the effect represents the inhibition of a mechanism for switching the locus of attention, although reference to earlier data recorded under similar experimental conditions broadens the scope for deliberation (Mountcastle er al., 1981, 1987). Since attention was not the chief subject of our study we refrain from contriving rival hypotheses whose evaluation would depend on experiments specifically addressed to that purpose. An extraretinal factor that is much better controlled experimentally is the position of the eye in the orbit, or the direction of gaze. Cells influenced by this factor are somewhat widespread in the visual cortex. We have found them in both V6 and V6A (Galletti et al., 1991a, 199.5). to add to their known distribution through areas V3A, LIP, 7a and MST (Andersen and Mountcastle, 1983; Galletti and Battaglini, 1989; Andersen ef al., 1990b; Squatrito ef al., 1994). More specific to V6A seems to be a class of neuron that we have previously termed real-position cells (Galletti et al., 1993). and which may utilize the output of gaze-modulated neurons (Galletti et al., 1995). Real-position cells have a receptive field organized in craniotopic, not retinotopic coordinates, and are likely involved in signalling the location of an object in space. These neurons are few in number, but none has been found in V6 and we believe that they may provide a key to the evolution of functional character that takes place between V6 and V6A. The functional role of V6 and V6A Briefly, we imagine from the rich anatomical connections between V6 and V6A and neighbouring parts of somatosensory area 5 (Colby et al., 1988; S. Shipp, M. Blanton and S. Zeki, manuscript in preparation) and with the premotor cortex (S. Shipp, M. Blanton and S. Zeki, manuscript in preparation), that V6N6A is a prominent source of the visual information that subserves visuomotor integration. If so, much of this information must concern the location of external objects in three-dimensional space, or in relation to the body; and thus to perform such a role V6 and V6A must undertake a coordinate transformation from their essentially retinotopic input. Real-position cells offer concrete evidence for this proposition. Other evidence might concern selectivity for location in depth, using stereo, ocular convergence or other cues. Some examples are already known for other areas of the parietal cortex. The fixation neurons described by Sakata et al. (1980, 1985) were sensitive to the location of gaze in three-dimensional space. And in area VIP, Colby et al. (1993) have recently described non-stereo, depth-specific responses that can only be elicited from a display screen placed very near to the animal. It is quite plausible that a number of our non-visual neurons in V6A were similarly selective for extreme stimulus proximity, and totally unresponsive to stimulation at a distance of 57 cm from the animal s face, which was invariant in our experiments. Another relevant factor is the input of somatosensory signals to V6 and V6A, which might generate bimodal responsiveness. Bimodal neurons have been described in areas MIP and VIP (Colby and Duhamel, 1991), nearby areas in the intraparietal sulcus that are both interconnected with PON6 and V6A (Colby et al., 1988; S. Shipp, M. Blanton and S. Zeki, manuscript in preparation). These bimodal neurons generally have visual and somatosensory receptive fields that are congruent with respect to the location or trajectory of their preferred stimuli in extrapersonal space. They may respond independ- ently to either modality or display various degrees of intramodal facilitation. Strong bimodal facilitation provides another possibility to account for the unresponsiveness of our non-visual neurons in V6A, which were only specifically tested with visual stimuli. It is also worth considering that the provision of a somatosensory cue might play a developmental role in helping to fix the location of the receptive field of a real-position cell that is also bimodal. Hence real-position cells might only be found in bimodal, but not purely visual areas. Visual responses that appear to be organized in head- or bodycentred coordinates have also been reported in areas of premotor cortex, where they appear as part of a complex response profile that also involves somatosensory and motor components (Gentilucci et al., 1983; Fogassi et al., 1992; Graziano et al., 1994b). The premotor cortex has few known direct links with the visual cortex (Kunzle, 1978; Godschalk et al., 1984; Matelli et al., 1986), so that its connection to V6A is clearly of interest (S. Shipp, M. Blanton and S. Zeki, manuscript in preparation). The anatomical input overlaps with a region in which premotor arm-related neurons have been described (Caminiti et al., 1991), suggestive of a fairly direct role in visuomotor coordination: the output from V6A might supply the premotor cortex with the visuo-spatial information required for the visual control of arm-reaching movements (Galletti et al., 1995). Acknowledgements We are grateful to L. Sabattini, G. Mancinelli, S. Boninsegna, B. Cotsell and G. Wray for technical assistance, J. Romaya for statistical software, and Chemical Industries Bracco S.p.A. for supplying the neurosurgical cement. This work was supported by EC grant CHRX-CT and by grants from Minister0 dell universita e della Ricerca Scientifica e Tecnologia and Consiglio Nazionale delle Ricerche (Bologna, Italy) and from the Wellcome Trust (London, UK). Abbreviations LIP MIP MST MT PO Pod POS SPG VIP WGA- -HRP References lateral intraparietal area medial intraparietal area medial superior temporal area middle temporal area parieto-occipital area dorsal parieto-occipital area parieto-occipital sulcus superior parietal gym ventral intraparietal area wheatgerm agglutinin conjugated to horseradish peroxidase Andersen, R. A. and Mountcastle, V. B. (1983) The influence of the angle of gaze upon the excitability of the light-sensitive neurons of the posterior parietal cortex. J. Neurosci., 3, Andersen, R. 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