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The Occipitoparietal Pathway of the Macaque Monkey: Comparison of Pyramidal Cell Morphology in Layer III of Functionally Related Cortical Visual Areas Guy N. Elston and Marcello G. P. Rosa The dendritic morphology of pyramidal cells located at the base of layer III in the primary visual area (V1), the second visual area (V2), the middle temporal area (MT), the ventral portion of the lateral intraparietal area (LIPv) and in the portion of cytoarchitectonic area 7a within the anterior bank of the superior temporal sulcus was revealed by injecting neurons with Lucifer Yellow in fixed, flattened slices of macaque monkey visual cortex. These areas correspond to different levels of the occipitoparietal cortical ‘stream’, which processes information related to motion and spatial relationships in the visual field. The tissue was immunocytochemically processed to obtain a light-stable diaminobenzidine reaction product, revealing the dendritic morphology in fine detail. Retrogradely labelled MT-projecting neurons in supragranular V1 (layer IIIc of Hassler’s nomenclature, corresponding to Brodmann’s layer IVb) were predominantly pyramidal, although many spiny multipolar (stellate) cells were also found. The average basal dendritic field area of pyramidal neurons in sublamina IIIc of V1 was significantly smaller than that in the homologous layer of V2, within the cytochrome oxidase-rich thick stripes. Furthermore, the average basal dendritic field areas of V1 and V2 pyramidal neurons were significantly smaller than those of neurons in MT, LIPv and area 7a. There was no difference in basal dendritic field area between layer III pyramidal neurons in areas MT, LIPv and 7a. While the shape of most basal dendritic fields was circularly symmetrical in the dimension tangential to the cortical layers, there were significant biases in complexity, with dendritic branches tending to cluster along particular axes. Sholl analysis revealed that the dendritic fields of neurons in areas MT, LIPv and 7a were significantly more complex (i.e. had a larger number of branches) than those of V1 or V2 neurons. Analysis of basal dendritic spine densities revealed regional variations along the dendrites, with peak densities being observed 40–130 µm from the cell body, depending on the visual area. The peak spine density of layer III pyramidal neurons in V1 was lower than that observed in V2, MT or LIPv, which were all similar. Pyramidal neurons in area 7a had the greatest peak spine density, which was on average 1.7 times that found in V1. Calculations based on the average spine density and number of dendritic branches at different distances from the cell body demonstrated a serial increase in the total number of basal dendritic spines per neuron at successive stations of the occipitoparietal pathway. Our observations, comparing dendritic fields of neurons in the homologous cortical layer at different levels of a physiologically defined ‘stream’, indicate changes in pyramidal cell morphology between functionally related areas. The relatively large, complex, spine-dense dendritic fields of layer III pyramidal cells in rostral areas of the occipitoparietal pathway allow these cells to sample a greater number of more diverse inputs in comparison with cells in ‘lower’ areas of the proposed hierarchy. visual areas located in the parietal and temporal lobes. Connections between areas in these two pathways are lamina-specific, and contrasting patterns of laminar origin and termination of pathways have been used to arrange visual areas into anatomical hierarchies (Rockland and Pandya, 1979; Wong-Riley, 1979a; Tigges et al., 1981; Maunsell and Van Essen, 1983a; Colby et al., 1988; Andersen et al., 1990; Boussaoud et al., 1990; Sousa et al., 1991; Rosa et al., 1993). Projections that originate mainly in the supragranular layers and terminate around layer IV are referred to as ‘feedforward’, and projections that originate mainly from infragranular layers and terminate outside layer IV are referred to as ‘feedback’ (e.g. Rockland and Pandya, 1979; Maunsell and van Essen, 1983a). Thus, V1 is at the ‘low’ end of the proposed hierarchy, and the visual areas of the temporal and parietal lobes are at the ‘high’ end (for review see Felleman and Van Essen, 1991). Basal dendritic territories of pyramidal neurons at an ‘intermediate’ level of the occipitotemporal pathway (the fourth visual area, V4) are larger than those in ‘lower’ stations such as V1 and the second visual area (V2; Lund et al., 1993). In addition, recent studies in macaque have shown an increase in the cross-sectional area of axonal clusters of intrinsic connections along the proposed hierarchy of visual areas, from V1 to parietal area 7a and the inferior temporal cortex (Yoshioka et al., 1992; Amir et al., 1993; Lund et al., 1993; Fujita and Fujita, 1996). This trend has been interpreted as a correlate of the anatomical hierarchy of processing, whereby the more widespread intrinsic connections, coupled with the larger centre to centre spacing, would ref lect the ‘more global computational roles proposed for higher-order visual areas’ (Amir et al., 1993). In parallel with these obser vations, Lund and co-workers (1993) also demonstrated that there is a proportional relationship between the cross-sectional area of intrinsic axonal clusters and the dendritic spread of supragranular pyramidal neurons in cortical areas: the larger the intrinsic axonal clusters in a given area, the larger the dendritic fields of pyramidal neurons. In view of these results, one might expect the dendritic field areas of pyramidal neurons to increase progressively along the pathways that link V1 to the parietal and temporal lobes. Indeed, in a recent study of marmoset monkey visual cortex (Elston et al., 1996), we found a progressive increase in the dendritic field area of layer III pyramidal cells between V1, V2, the dorsolateral area (DL) and the fundus of the superior temporal area (FST). Although these observations could be interpreted as supporting the anatomicalhierarchical model, the evidence is still inconclusive, mainly because comparisons have been made between areas belonging to the occipitoparietal and occipitotemporal pathways, which process different modalities of vision. For example, both DL in the marmoset and its macaque homologue (V4) are linked to the processing of object shape and colour, whereas FST and area 7a process motion and spatial relationships (e.g. Boussaoud et al., The visual system of primates comprises multiple cortical areas, located in the occipital, temporal and parietal lobes. Two main cortical pathways have been proposed on the basis of functional and anatomical criteria, streaming from striate cortex (V1) into Vision, Touch and Hearing Research Centre, Department of Physiology and Pharmacology, The University of Queensland, Queensland 4072, Australia Cerebral Cortex Jul/Aug 1997;7:432–452; 1047–3211/97/$4.00 Table 1 Animals and visual areas in which neurons were injected Animal Age (months) Sex Weight (kg) Areas studied Plane of sectioning AM 1 RM 13 RM 14 RM 16 14 20 20 96 male male female female 1.3 1.5 1.5 2.2 V2 V1, 7a MT, LIPv V1 tangential tangential tangential coronal 1990; Distler et al., 1993). Furthermore, previous studies did not establish whether the neurons sampled in V1 and V2 were part of the occipitoparietal or occipitotemporal pathways. We addressed this problem by investigating the dendritic fields of pyramidal neurons in striate cortex and four extrastriate areas that correspond to different levels of the proposed motionprocessing pathway of the macaque visual system. Pyramidal cells located at the base of layer III in V1 (layer IVb of Brodmann), the origin of the occipitoparietal pathway, were compared with basal layer III pyramidal neurons located within the thick cytochrome oxidase-rich stripes of the second visual area (V2), in the middle temporal area (MT), in the ventral portion of the lateral intraparietal area (LIPv; Blatt et al., 1990) and in the portion of cytoarchitectonic area 7a located in the dorsal superior temporal sulcus (Hikosaka et al., 1988; Andersen et al., 1990). This was achieved by intracellular injection of the naphthalamide tracer Lucifer Yellow in fixed cortical slices. These data also allowed the study of other aspects of basal dendritic field morphology in extrastriate cortex that have not been addressed by previous studies, such as the relative complexity of dendritic fields, dendritic biases and the spine density of neurons of different visual areas. Materials and Methods Animals and Tracer Injection Four macaque monkeys (Macaca fascicularis) were used in this study (Table 1). The monkeys were used in non-related acute experiments involving electrophysiological recording or optical imaging, under pentobarbitone sodium anaesthesia (Calford et al., 1995). Three of these cases (AM 1, RM 13 and RM 14), which yielded the bulk of the data, were juveniles (14–20 months old). In a fourth, 8-year-old animal (RM 16), a 1 µl injection of the f luorescent tracer Fluoroemerald (7% in H2O; Molecular Probes D 1820) was placed in area MT at the beginning of a non-related 3 day electrophysiological recording experiment, in order to label neurons retrogradely in V1. In this case, after initial anaesthesia (ketamine, 50 mg/kg and xylazine, 6 mg/kg; pentobarbitone sodium, 7 mg/kg), the animal was placed in a stereotaxic frame and the skull overlying the superior temporal and lateral sulci was removed. The injection was made with the aid of a 1 µl microsyringe (Scientific Glass Engineering, Australia) mounted in a stereotaxic manipulator. The microsyringe was lowered into the cortex from the surface of the anterior bank of the superior temporal sulcus to a depth of 5 mm (Fig. 1A). This anterior–posterior parasagittal approach resulted in penetration of the posterior bank, including area MT. A series of 200 nl injections of Fluoroemerald was made at 3–5 min intervals at slightly different points along the track, to make a total of 1 µl. The microsyringe was left in place for 10 min after the final injection, then retracted. The resulting injection site was centred on area MT (Fig. 1B), but also leaked into the underlying white matter and anterior bank of the superior temporal sulcus, probably involving the medial superior temporal area (MST; Maunsell and Van Essen, 1983a). As no projection from V1 to MST has been demonstrated, this did not affect our results. Histological Processing and Identification of Visual Areas Upon completion of the electrophysiological experiments, the animals were administered an i.v. injection of 200 mg/kg of sodium Figure 1. Schematic showing (A) the approach used for the injection of Fluoroemerald in animal RM16. The injection syringe penetrated via the anterior bank of the superior temporal sulcus, but deposited the tracer mainly in the posterior bank of this sulcus. Serial reconstruction of coronal sections revealed the core of the injection was in the middle temporal area (MT). Two coronal sections (B) in which the injection site was clearly visible are illustrated. Scale bar = 1 cm. pentobarbitone. They were then perfused intracardially with normal saline, followed by 4% paraformaldehyde (in 0.1 M phosphate buffer, pH 7.2). Blocks of tissue (Fig. 2) were excised from the occipital operculum (V1), the posterior bank of the inferior occipital sulcus (V2), the posterior and anterior banks of the dorsal superior temporal sulcus (including areas MT and 7a) and the lateral bank of the intraparietal sulcus (including area LIPv). Only the hemispheres contralateral to those involved in electrophysiological recording experiments were used for intracellular injection. Identification of areas MT, LIPv and 7a within the slices (Fig. 2) was based on the sulcal morphology, using maps derived from previously published studies (Gattass and Gross, 1981; Van Essen et al., 1981; Desimone and Ungerleider, 1986; Andersen et al., 1990; Blatt et al., 1990; Boussaoud et al., 1990; Shipp and Zeki, 1995). We were particularly careful to avoid the regions around the caudal lip of the superior temporal sulcus, which include parts of areas V4 and V4t, and around the lips and fundus of the intraparietal sulcus, which include the dorsal subdivision of LIP and the ventral intraparietal area respectively (Schein et al., 1982; Maunsell and Van Essen 1983a; Gattass et al., 1988; Blatt et al., 1990; Colby et al., 1993). The ventral portion of LIP, which receives input from MT (Desimone and Ungerleider, 1986; Blatt et al., 1990; Shipp and Zeki, 1995), was selected for injection, as opposed to the dorsal portion which receives input from both TEO and TE (Webster et al., 1994; Shipp and Zeki, 1995). The blocks were f lattened between glass slides (e.g. Rosa et al., 1988), left overnight in fixative (4% paraformaldehyde in 0.1 M phosphate buffer) and then sectioned on a Vibratome. The exception was animal RM 16, whose cortex was sectioned coronally in order to allow reconstruction of the Fluoroemerald injection site in area MT (Fig. 1B) and the morphological classification of retrogradely labelled cells in a more traditional plane of sectioning. Alternate 50 and 250 µm slices were obtained from the f lat-mounted blocks of tissue including V1 and V2, the Cerebral Cortex Jul/Aug 1997, V 7 N 5 433 Figure 2. Schematics showing the parts of the brain from which blocks of tissue were excised (grey, in the inserts) and the relative location of individually injected neurons (dots) in flat-mounted slices obtained from areas V1, V2, MT, LIPv and 7a. In the V2 diagram the occipital operculum was everted, revealing the posterior banks of the lunate and inferior occipital sulci. The magnified view of the posterior bank of the inferior occipital sulcus indicates the location of cytochrome oxidase-rich thick stripes (dark grey) where MT-projecting neurons are located, thin stripes (light grey) and cytochrome oxidase-poor interstripes (white). In the MT diagram the approximate borders of this area are indicated, based on the heavier myelin content, which is visible even in unstained tissue. The scale bar (under the V2 diagram) corresponds to 5 mm. 50 µm slices being reacted for cytochrome oxidase (Wong-Riley, 1979b) to reveal laminar features, the ‘blobs’ of V1 and the ‘stripes’ of V2 (Horton and Hubel, 1981; Tootell et al., 1983). Only 250 µm slices were obtained from the other areas, and the identification of layers was based on cytoarchitecture (see below). Nomenclature of Cortical Layers Throughout this paper the nomenclature of cortical layers follows the scheme proposed by Hassler (1966) rather than that proposed for macaque striate cortex by Brodmann (1909). The main differences between Hassler’s and Brodmann’s nomenclatures are summarized in Table 2. We have decided to use the Hassler scheme because, based on cytology and efferent connections, there is now ample evidence to support the view that some of the subdivisions of Brodmann’s layer IV (layers IVa and IVb) are more accurately described as subdivisions of layer III (for a review see Casagrande and Kaas, 1994). Unlike the true granular layer of primary sensory cortex, Brodmann’s sublamina IVb contains many pyramidal neurons which provide direct ‘feedforward’ input to other cortical areas in both the ipsi- and contralateral hemispheres (Lund 434 Supragranular Pyramidal Cells in Visual Cortex • Elston et al. Table 2 Comparison between the nomenclature for the cortical layers of primate V1 used in the present study (Hassler, 1966) and the one proposed by Brodmann Hassler Brodmann (1909, tr. Garey 1994) I II IIIa IIIb IIIc IVα IVβ V VI I II III IVa IVb IVcα IVcβ V VI et al., 1975; Rockland and Pandya, 1981; Maunsell and Van Essen, 1983a; Kennedy et al., 1986; Shipp and Zeki, 1989a; present results). Thus, Brodmann’s sublamina IVb is more properly regarded as a subdivision of layer III, as made explicit by Hassler’s nomenclature (IIIc). All of our sample, in different areas, comes from supragranular cortex just above the upper limit of Hassler’s layer IV, and this nomenclature emphasizes the fact that our analyses involved comparable populations of cells in the different areas. Furthermore, our laboratory is also involved in studies in various New World monkey species, for which the Hassler (1966) nomenclature of cortical layers is usually employed; thus, we opted for the use of a consistent nomenclature between studies. Identification of Layer III In case RM 16 we specifically targeted neurons in transverse sections in V1 that were retrogradely labelled from area MT, and therefore known to be located in sublamina IIIc (Shipp and Zeki, 1989a; Vogt Weisenhorn et al., 1995). The remaining cases were sectioned tangential to the cortical layers. In V1 we identified the upper limit of sublamina IIIc in f lat-mounted sections by cytochrome oxidase patterns, using the characteristic cytochrome oxidase ‘honeycomb’ of sublamina IIIb as a criterion (Hendrickson et al., 1978). Furthermore, in all areas the basal limit of layer III was determined by the distinct cytoarchitecture of layers III and IV. To determine cytoarchitecture, the slices were incubated for 10 min in 10–5 M 4,6-diamidino-2-phenylindole (DAPI; Sigma D9542) in 0.1 M phosphate buffer, which enabled the visualization of somata by f luorescence microscopy. By focusing through the thickness of the slice, we found that layer III was characterized by relatively large, sparsely spaced somata of pyramidal cells, whereas layer IV contained the relatively small, closely packed cell bodies of granule cells (Fig. 3). Furthermore, injection of the densely packed DAPI-labelled neurons below the transition point yielded only the small multipolar neurons characteristic of the granular layer (unpublished results). In V2 only layer III neurons whose somata were located within the thick cytochrome oxidase-rich stripes (Tootell et al., 1983; Livingstone and Hubel, 1987; Shipp and Zeki, 1985, 1989b) were included for analysis. Similar to other studies using appropriately f lattened sections of V2 (e.g. Shipp and Zeki, 1989b; Abel et al., 1997), we found that the morphological distinction between thick and thin cytochrome oxidase-rich stripes was relatively obvious. Intracellular Injection Pre-labelling the tissue with DAPI facilitated rapid impalement of a large number of cells in a single slice. Neurons in layer III were injected in a semiregular array, which minimized any possible effects of experimenter bias. The injection apparatus consisted of a fixed stage Zeiss microscope, equipped with a ×40 long focal length immersion lens, a Leitz micromanipulator and a perspex tissue chamber mounted on the stage. The tissue was immersed in 0.1 M phosphate-buffered saline (pH 7.2). The slices were mounted such that the layer closest to the pia mater was uppermost in the injection chamber. Somata within ∼100 µm of the surface of the tissue, but superficial to the layer III/ IV interface, were injected to reveal a short portion of the apical dendrite and the complete extent of the basal skirt of dendrites. A similar technique has been employed in previous studies addressing tangential dendritic field areas of pyramidal cells in sensory cortex (Hübener and Bolz, 1992; Matsubara et al., 1996; Elston et al., 1996, 1997). Micropipettes (250–300 MΩ) were pulled on a Sutter microelectrode puller (Brown Flaming P77, San Francisco, CA). Individual neurons were injected with Lucifer Yellow (Sigma L 0259; 8% in 0.05 M Tris buffer) by hyperpolarizing current (a maximum of 100 nA for 10–20 s, subject to visual determination). Following cell injection, the tissue was processed with an antibody to Lucifer Yellow (kindly supplied by Dr David Pow) at a concentration of Figure 3. (A) Photomicrograph showing a cross-section of extrastriate cortex that was labelled with DAPI (4,6-diamidino-2-phenylindole), as viewed with UV excitation (341–343 nm). (B, C) Sections through layers III (B) and IV (C) of flat-mounted tissue. Note that in both cross-sectional and flattened preparations the granular layer was easily distinguished from the supragranular layer due to the large number of closely spaced neurons with relatively small somata. Scale bar (in C) = 200 µm for (A) and 75 µm for (B) and (C). The arrow in (A) points to the upper limit of layer IV. Cerebral Cortex Jul/Aug 1997, V 7 N 5 435 1:400 000 in stock solution [2% bovine serum albumin (Sigma A3425), 1% Triton X-100 (BDH 30632) and 5% sucrose in 0.1 M phosphate buffer]. The primary antibody was detected by a species-specific biotinylated secondary antibody (Amersham RPN 1004; 1:200 in stock solution for 2 h) then serially processed with a biotin–horseradish peroxidase complex (Amersham RPN 1051; 1:200 in 0.1 M phosphate buffer). Labelling was revealed using 3,3′-diaminobenzidine (DAB; Sigma D 8001; 1:200 in 0.1 M phosphate buffer) as a chromagen (Fig. 4). Fluoroemerald injection in MT, and 428 DAPI-labelled neurons in areas V1, V2, MT, LIPv and 7a. Of these, 41 retrogradely labelled V1 cells were successfully reconstructed in transverse sections to a level of detail that allowed their classification as pyramidal or multipolar, and 183 DAPI-labelled pyramidal cells in f lat-mounted tissue were successfully filled as per the three criteria specified in Materials and Methods. Analysis Injected cells were classified as pyramidal or multipolar (Lund, 1984; Nieuwenhuys, 1994; Elston et al., 1996), and were drawn with the aid of a camera lucida. In order to be included for further analysis injected neurons in f lattened sections had to satisfy three criteria: (i) they had to be located at the base of layer III; (ii) they had to be unambiguously identified as pyramidal cells by the presence of the characteristic apical dendrite, which could be visualized by focusing through the thickness of the slice (see fig. 3 of Elston et al., 1996); and (iii) they had to be successfully filled, thus revealing their complete basal dendritic arborization as well as fine details such as dendritic spines (Fig. 4). The applicability of the last criterion to individual cells was judged by tracing each terminal dendrite to an abrupt, well defined tip which usually had a spinous process (see Matsubara et al., 1996). Cells in which the distal branches had an uneven or beaded appearance were excluded. The serendipitous nature of the slice boundaries relative to the cortical layers, and the desire to inject cells that were both away from the upper limit of the slice and above the limit of layer IV, meant that we were not able to obtain samples from each area in each monkey (Table 1). Instead, we concentrated the analyses in the best slice obtained from each area across cases, as judged by the appropriate laminar position of cells and quality of the cell filling. Note, however, that in every case in which we had more than one sample, they were not distinguishable in terms of morphological parameters such as dendritic field area. In f lattened sections, basal dendritic field areas were estimated by scanning the original drawings of the cells and drawing a polygon (hull) joining the outermost distal tips of each terminal dendrite (examples of drawn polygons are illustrated in Fig. 11). The area of the resulting polygon was then calculated using standard features of NIH-IMAGE (NIH Research Services, Bethesda, MD), which was run on a Macintosh Quadra 950 computer (Apple Computer Inc., Cupertino, CA). Other analyses of basal dendritic fields were performed on a sample of 20 neurons from each area, including cells whose dendritic fields ranged from the smallest to the largest (explained in detail in the Results section). These included a Sholl analysis (cf. Hübener and Bolz, 1992) aimed at comparing the complexity of the branching pattern in different areas (estimated by the number of intersections between dendritic branches and concentric circles of increasing radius centred on the cell body). We chose to use equal sample sizes rather than investigating each cell in each area, in order to improve the reliability of multifactorial ANOVA design employed in this analysis. The same sample was studied to explore the possibility of significant directional asymmetries in the distribution of dendritic branches, by determining the distribution of intersections as a function of the radial position of the dendrite relative to the cell body. Spine densities were calculated by drawing a selection of 20 basal dendrites of pyramidal neurons from each area at high power (using a Zeiss ×100 immersion objective and an Axioplan microscope). Because of the nature of the immunocytochemical stain employed, spines were visible by focusing across the dendritic shafts when viewed by high power light microscopy. Thus, correction factors for hidden spines, which may be needed for more opaque Golgi-stained material (Feldman and Peters, 1979), were deemed unnecessary. No distinction was made between different spine types, such as sessile or pedunculated (Jones and Powell, 1969). Cell bodies were also drawn at high power in order to test for differences between peak cell body cross-sectional areas, and for a correlation between cell body size and basal dendritic field area. Again, areas were calculated using standard features of the NIH-IMAGE software. Neurons in V1 That Project to MT The main objective of this study was to compare morphological features of a single cell type (pyramidal) in different visual areas that comprise the occipitoparietal pathway. In V1, specific populations of neurons project from layer IIIc to area MT (Tigges et al., 1981; Shipp and Zeki, 1989a). However, there is confusion as to whether pyramidal neurons in layer IIIc contribute to these projections. Thus, because we intended to compare neurons at different levels of the occipitoparietal pathway, it was first necessary to ascertain whether the study of pyramidal cells in layer IIIc of V1 was appropriate. The injection of the tracer Fluoroemerald in MT revealed retrograde labelling in V1 that extended from the medial portion of the occipital operculum to the dorsal bank of the calcarine sulcus [corresponding to the representation of eccentricities between 5 and 30° of the lower visual field (Daniel and Whitteridge, 1961)]. A ll of the retrogradely labelled neurons were located at the base of layer III (sublamina IIIc of Hassler, 1966). Of 41 MT-projecting cells that were successfully reconstructed, 28 (68.3%) were pyramidal (including modified pyramidal) and 13 (31.7%) were spiny multipolar neurons (Figs 5–7). Thus, pyramidal cells in layer IIIc form a substantial portion of the MT-projecting population. Pyramidal neurons were distinguished from spiny multipolar neurons by the presence of an unambiguous apical dendrite and a characteristic ‘skirt’ of basal dendrites (Figs 5B, D and 6). Spiny multipolar neurons lacked an apical dendrite, and their dendrites projected radially from the cell body (Figs 5C and 7B–D). Two types of spiny multipolar cells were retrogradely labelled from MT: one with a relatively small soma and relatively thin dendrites (Fig. 7C) and another with a very large cell body and relatively thick, wide ranging dendrites (Figs 5C and 7B–D). A number of neurons that were retrogradely labelled and subsequently injected had morphological characteristics intermediate between those of pyramidal and multipolar neurons. For example, these cells had an ‘apical’ dendrite with daughter segments which in many cases ref lected back toward the infragranular layers, and, instead of the characteristic ‘skirt’ of basal dendrites, they had multiple dendritic processes issuing from the soma in a radiate fashion (Fig. 7A). Such modified pyramidal neurons have been reported variously in sensory cortex (Lorente de No, 1938; Lund, 1973, 1984; Jones, 1975; Simons and Woolsey, 1984; Nieuwenhuys, 1994). In many of the injected neurons the initial part of the axonal trajectory was visible (Figs 6 and 7). In all cases where the axon could be visualized, it issued from the surface of the soma closest to the white matter. In most pyramidal neurons in which the axon was visible, the main segment, which projected directly toward the white matter, gave rise to horizontal collaterals within layer III (e.g. Fig. 7A). Results Intracellular injection of Lucifer Yellow was performed on 47 neurons in V1 that were retrogradely labelled from a 436 Supragranular Pyramidal Cells in Visual Cortex • Elston et al. Basal Dendritic Fields of Layer III Pyramidal Cells Our conclusions related to the tangential area of basal dendritic fields of pyramidal neurons, measured in f lat-mounted slices of five visual areas, are summarized in Figures 8 and 9. The main Cerebral Cortex Jul/Aug 1997, V 7 N 5 437 point revealed by this analysis can be qualitatively assessed simply by observing a sample of injected pyramidal neurons in different visual areas (Fig. 8): the basal dendritic fields of V1 and V2 neurons were generally smaller than those of cells in MT, LIPv and area 7a, but there was no obvious difference in area between dendritic fields of cells in the last three visual areas. In order to illustrate this point, cells in each area were ranked in ascending order of tangential basal dendritic field area. Cells corresponding to equivalent percentiles of the distributions of dendritic field area were then drawn, with dendritic fields collapsed to a single plane (Fig. 8). Figure 9 and Tables 3 and 4 provide graphic and statistical summaries of the entire data set. A one-way analysis of variance (Table 4) revealed a highly significant difference between areas (P < 0.0001). In spite of a large overlap between the basal dendritic field areas of pyramidal neurons located in V1 and in the thick stripes of V2 (Fig. 9), there was a statistically Figure 5. Photomicrographs illustrating Fluoroemerald-labelled MT-projecting neurons in sublamina IIIc of V1 (A) and neurons that were subsequently injected with Lucifer Yellow (B–D). Cells B and D have an apical dendrite characteristic of pyramidal neurons. Cell C, which lacks an apical dendrite, was classified as a spiny multipolar neuron by virtue of the radiate fashion in which dendrites issue from its soma (note the exceptionally large soma). Scale bar = 200 µm for (A) and 100 µm for (B–D). Figure 4. Photomicrographs of three extrastriate pyramidal neurons (A–C) located at the base of layer III, injected with Lucifer Yellow and processed for a DAB (3,3′-diaminobenzidine) reaction product. The plane of focus includes the cell body and the initial segments of the basal dendrites. The apical dendrite and distal tips of dendrites could only be visualized by focusing through the thickness of the section (not shown). Dendritic spines were clearly outlined, even at low power. There is little leakage of dye around the cell body, thus allowing the unambiguous determination of somal area and number of primary dendrites. (D) is a magnified view of (C), illustrating the dendritic spines in greater detail. Scale bar = 50 µm in (A–C), 20 µm in (D). 438 Supragranular Pyramidal Cells in Visual Cortex • Elston et al. Figure 6. (A–D) Drawings of four MT-projecting neurons located in sublamina IIIc of V1. These cells had the characteristic morphology of pyramidal neurons. Part of the axon was revealed and is illustrated in fine ink. Inserts show the position of cells relative to the pial surface (solid line) and the top of layer IV (dashed line). Scale bar = 100 µm. Cerebral Cortex Jul/Aug 1997, V 7 N 5 439 Figure 7. Drawings of MT-projecting V1 neurons. Cell (A) was classified as a modified pyramidal neuron due to the ambiguous ‘apical’ dendrite, the daughter segments of which project basally, and to the fact that the remaining dendrites radiated from the cell body in all directions, rather than forming the classical basal ‘skirt’. Neurons (B–D) were all classified as spiny multipolar (stellate). Two types of multipolar neurons project to area MT, one with a very large cell body and thick, long-ranging dendrites (B and D), the other with a relatively small cell body and restricted dendritic field (C). Axons are illustrated in fine ink. Inserts show the position of the cell relative to the pial surface (solid line) and the top of layer IV (dashed line). Scale bar = 100 µm. significant difference (P < 0.05) between the populations in these two areas (Table 4). The distributions of dendritic field area of cells in MT, LIPv and area 7a were clearly different from 440 Supragranular Pyramidal Cells in Visual Cortex • Elston et al. those observed in V1 and V2 (Figs 8 and 9), a result which was confirmed to be statistically significant (Tables 3 and 4). Finally, there were no significant differences in basal dendritic field area Figure 8. Basal layer III pyramidal neurons from V1, V2 (cytochrome oxidase- rich thick stripes), MT, LIPv and area 7a, viewed in the plane of section tangential to the cortical layers. The neuron with the smallest basal dendritic field area from each visual area is illustrated on the left of each row; the one with the largest is on the right of each row. The other neurons in each row represent 20% rank increments along the distribution of dendritic field area (i.e. the 20th, 40th, 60th and 80th percentiles). Scale bar = 100 µm. of pyramidal neurons located at the base of layer III between areas MT, LIPv and 7a (P > 0.05 in all cross-comparisons; see Table 4). In summary, there is evidence for a stepwise increase in dendritic field area of layer III pyramidal neurons at early stages of the proposed cortical hierarchy (V1, V2 and MT), but not between MT, LIPv and 7a. Complexity of the Branching Patterns A study of Figure 8 suggests that layer III pyramidal neurons in rostral visual areas may have more complex basal dendritic branching patterns than those in V1. However, a comparison of the number of primary dendrites issuing from the cell body showed no significant difference between areas (not illustrated). In order to investigate this issue quantitatively, we performed a Sholl analysis on the dendritic fields of cells from each area. The result of this analysis (Fig. 10) confirmed that pyramidal cells in layer III of all areas studied had similar branching patterns near the cell body (in a ring 25 µm away from the centre). However, the overall pattern of branching complexity was significantly different between areas (Table 5). Cells in V1 and V2 had very similar branching patterns throughout, both quantitatively and qualitatively. In both areas the number of intersections peaked in a relatively narrow region ∼50 µm from the centre of the cell body, and then gradually decreased with very few branches reaching beyond 150 µm. This pattern contrasts with the one observed in areas MT, LIPv and 7a, where the peak number of intersections occupied a broader region, ∼50 µm wide, centred around 75 µm from the cell body. As shown in Figure 10, the complexity of the dendritic tree in these areas, evaluated by the number of intersections at equivalent distances from the cell body, was greater than in V1 and V2. This conclusion is true even if a comparison is restricted to the region where maximal complexity was found in V1 and V2 (50 µm ring). Finally, while the higher number of intersections suggested that the dendritic trees in LIPv were more complex than those in either MT or area 7a (Fig. 10), this result was not statistically significant (Table 5). Cerebral Cortex Jul/Aug 1997, V 7 N 5 441 Table 3 Basal dendritic field areas of pyramidal neurons in deep layer III Visual area n Mean SD SEM Minimum Maximum (× 103 µm2) (× 103 µm2) (× 103 µm2) (× 103 µm2) (× 103 µm2) V1 V2 MT LIPv 7a 25 45 34 40 39 35.8 44.8 83.8 88.1 84.7 5.5 10.4 10.6 16.0 23.3 1.10 1.55 1.82 2.53 3.74 27.3 17.8 56.1 60.0 37.2 48.5 65.8 104.2 149.6 145.5 Table 4 One-way ANOVA: dendritic field area of neurons in different visual areas Source Sum of squares 10 Between 8.55 × 10 areas Within areas 3.99 × 1010 Total 1.25 × 1011 DF 4 178 182 Mean squares 10 2.14 × 10 F-ratio Probability 95.34 <0.0001 8 2.24 × 10 One-tailed between-groups comparison: Dunnett’s t procedure V2 MT LIPv 7a V1 V2 MT LIPv 2.42* 12.18** 13.70** 12.75** 11.46** 13.29** 12.17** 1.22 0.25 1.00 *P < 0.05; **P < 0.01. Figure 9. Frequency histograms of basal dendritic field areas of layer III pyramidal neurons in V1, V2, MT, LIPv and area 7a in the macaque monkey. Basal dendritic fields of layer III pyramidal neurons in V1 were smaller than those of V2, which were smaller than those of cells in MT. There was no significant difference in basal dendritic field area of layer III pyramidal neurons between areas MT, LIPv and 7a. Arrows indicate the means for each area. 442 Supragranular Pyramidal Cells in Visual Cortex • Elston et al. Dendritic Field Asymmetries We also investigated whether basal dendrites of pyramidal cells were symmetrically arranged around the cell body or demonstrated bias towards particular axes. The vast majority of the dendritic trees were circular or gently elliptical, with the cell body occupying the approximate centre. Few cells had somata that were obviously eccentric relative to the tangential extent of the dendritic trees (e.g. in area 7a; see the bottom row of cells in Fig. 8). However, a closer inspection of the data also suggested that, in some cases, dendritic branches tended to cluster in some directions relative to the cell body, even though this did not result in an overall elongation of the dendritic field. To evaluate this possibility, we subdivided the counts of intersections used for the Sholl analysis in arbitrary 30° polar angle sectors centred on the cell body, and generated polar plots (Fig. 11) that indicate the number of intersections in different directions (including cumulative data for distances ranging from 25 to 150 µm from the cell body). The result of this analysis (Fig. 12) demonstrates that dendritic biases existed, and that the nature of the bias varied in the different areas. In order to compare the dendritic biases in different visual areas we selected 20 neurons from each visual area, representing regular increments along the distributions of basal dendritic field area. We classified the polar plots of dendritic branches versus direction from the cell body in four categories. By analogy with the well-known polar plots of direction selectivity of neuronal responses (e.g. Maunsell and Van Essen, 1983b; Albright, 1984), dendritic fields of cells were classified as ‘morphologically orientation-biased’ if dendritic branches tended to cluster in two diametrically opposed directions away from the cell body. To fit this category, the counts of number of intersections in the preferred directions had to be similar (within 50%), and their sum had to be at least twice as large as that of the orthogonal directions (Fig. 11A). ‘Morphologically direction-biased’ Table 5 Two-way ANOVA, repeated measures (n = 20), on Sholl analysis (5 × 6 design) area (V1, V2, MT, LIPv, 7a) by distance from cell body (25, 50, 75, 100, 125, 150 µm) Source Sum of squares Mean squares F-ratio Probability (P) Area Distance Area × distance Within cells 13052.82 16605.07 3738.14 DF 4 5 20 3263.20 3321.01 186.91 158.61 161.42 9.08 <0.01 <0.01 <0.05 11726.81 570 20.57 Post-hoc comparison between pairs of areas (Newman–Keuls) V2 MT LIPv 7a V1 V2 MT LIPv n.s. P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05 n.s. n.s. n.s. degrees of clustering (Fig. 11B, C). Some neurons had dendrites that radiated more or less homogeneously in all directions from the cell body, forming a symmetrical polar plot. These cells were termed ‘nonbiased’ (Fig. 11E). Finally, a few cells could not be categorized according to the above criteria. For example, some neurons had two clusters of dendrites that were not diametrically opposed and could therefore not be classified as ‘morphologically orientation-biased’, and some cells had three clusters of dendrites radiating from the cell body (Fig. 11D). These cells were classified as ‘morphologically complex’. Analysis of the data revealed significant differences between cortical areas in the relative proportions of cells that fitted each category (Table 6). In V1 layer IIIc, but in no other area, a relatively large proportion of cells (45%) had morphologically orientation-biased dendritic fields (Fig. 12A). Another 40% of the V1 sample showed morphological direction-bias, mostly in a narrow range of directions (Fig. 12B). Only 15% of the V1 sample showed no directional clustering of dendrites. In sharp contrast, within the thick stripes of V2 a majority of cells (75%) had morphologically direction-biased dendritic fields (Fig. 12C), with the remaining cells showing either a mild morphological orientation-bias or no directional preference (Fig. 12D). No consistent relationship was detected between the axes of dendritic clustering in different cells and the visuotopic representations in V1 or V2. Morphologically orientation-biased cells were rare in the higher-order areas of the occipitoparietal pathway: only 10–20% of the cells sampled in MT, LIPv and area 7a showed any evidence of morphologically orientation-biased dendritic fields. In these three areas, the majority of neurons had either slightly morphologically direction-biased or nonbiased dendritic fields (Fig. 12E–H). Figure 10. Sholl analysis of dendritic trees of layer III pyramidal neurons in five visual areas of the visual occipitoparietal pathway. dendritic fields were those in which the dendritic branches clustered in a particular direction, resulting in an intersection count that was at least twice as large as that in the diametrically opposed direction. This category included cells with different Spine Densities Along Basal Dendrites Our analysis of dendritic spine density demonstrated a similar trend in all areas studied. Generally (Fig. 13), spine density was low within 40 µm of the cell body, reached a peak in the region 40–130 µm away from the cell body (according to the cortical area) and gradually decreased towards the distal tips. This variability meant that calculations of average spine density were not particularly meaningful, and we therefore decided to do statistical comparisons of the peak spine densities between areas. In order to do this we ascertained, from the graphs showing mean spine density as a function of distance from the cell body (Fig. 13), which combination of five consecutive 10 µm Cerebral Cortex Jul/Aug 1997, V 7 N 5 443 20 10 15 15 20 10 15 Figure 11. Classification of dendritic tree biases. Five examples of cells from areas MT, LIPv and 7a are illustrated on the left. The corresponding polar plots of dendritic tree intersections (from Sholl analysis) as a function of direction from the cell body are illustrated on the right. (A) Morphologically orientation-biased dendritic field; (B) narrowly morphologically direction-biased dendritic field; (C) broadly morphologically direction-biased dendritic fields; (D) morphologically complex dendritic field, with dendrites clustering in three non-diametrically opposed directions; (E) nonbiased dendritic field. The radial dimension in the polar plots was computed by adding the number of intersections with imaginary circles 25, 50, 75, 100, 125 and 150 µm from the cell body, in 30° polar angle intervals. Each division along the axes equals 5 intersections. The directions relative to the cell body are arbitrary. The basis of the classification is explained in the main text. The polygons around the cells (fine dashed line) illustrate the criterion for delimitation of basal dendritic field areas (e.g. Young and Vaney, 1991). Scale bar = 100 µm. 444 Supragranular Pyramidal Cells in Visual Cortex • Elston et al. segments of dendritic shaft yielded the highest spine count (arrowheads in Fig. 13), and calculated ‘peak spine density’ as the average of all counts within this 50 µm segment. We found, by means of a one-way analysis of variance and post-hoc analysis (Table 7), that the peak density of basal dendritic spines of neurons in V1 (mean ± SD: 5.79 ± 2.59 spines per 10 µm dendritic segment) was significantly lower than that in V2 (7.42 ± 2.09 spines/10 µm), that the peak spine density in V2 was not different from that in MT (7.95 ± 2.04 spines/10 µm) and LIPv (7.68 ± 2.06 spines/10 µm), and that the peak spine density in area 7a (9.83 ± 2.46 spines/10 µm) was greater than that in all other areas. The total number of spines in the basal dendritic field was calculated for the ‘average’ neuron in each visual area. In order to calculate this, the average number of dendritic branches at different distances from the cell body (in 25 µm increments), obtained from the Sholl analysis, was multiplied by the average number of spines along the corresponding 25 µm sector of dendritic shaft. Using this approximation, it was apparent that the increase in tangential area and complexity of dendritic fields, combined with the larger spine densities in rostral areas of the occipitoparietal pathway (as compared with V1), resulted in a gradual increase in the number of spines per neuron. While neurons in V1 had on average 799 spines along their basal dendritic trees, in V2 they had 1201 spines, in MT 2077 spines, in LIPv 2316 spines and in area 7a 2572 spines. Thus, although the peak spine density of area 7a neurons was 1.7 times that of neurons in V1, there was a 3.2-fold difference between the total number of spines in the basal dendritic fields of the ‘average’ neuron in these areas. Somal Areas of Layer III Pyramidal Cells The peak cross-sectional areas of cell bodies of pyramidal neurons were compared between areas, using the tangentially sectioned tissue. One hundred and seventy-seven of the 183 neurons were used for this analysis. Six neurons were excluded due to leakage of dye around the soma. A nonparametric test (Kruskal–Wallis) across the entire sample revealed significant differences between areas, as summarized in Table 8. Mann– Whitney U-tests demonstrated significant differences for all comparisons between pairs of areas, with the exception of the comparison between MT and area 7a (Fig. 14; Tables 8 and 9). There was no apparent correspondence between cell body size and position in the proposed anatomical hierarchy: somal areas of pyramidal neurons at the base of layer III in V1 were larger than those in the corresponding layer of V2, although there was a large overlap between populations, and areas of neurons in LIPv were larger than those of neurons in area 7a. When somal cross-sectional areas were compared with dendritic field areas of individual neurons, a significant relationship between these two variables was revealed (Fig. 15). Discussion The present study was designed to compare the morphology of a specific neuronal type (pyramidal cell) in the homologous layer (basal layer III) of areas that are known to be functionally interrelated in the occipitoparietal pathway. Pyramidal cells in layer III are the principal originators of ‘feedforward’ corticocortical projections, as well as participating in interhemispheric connections and, to a lesser extent, ‘feedback’ projections to other areas (for a review see Felleman and Van Essen, 1991). Basal dendritic fields of layer III pyramidal neurons in extrastriate areas MT, LIPv and 7a were compared with those in Figure 12. Examples of polar plots showing dendritic bias in different visual areas. The illustrated directions are arbitrary. (A) Five morphologically orientation-biased V1 dendritic fields; (B) five morphologically direction-biased V1 dendritic fields; (C) five morphologically direction-biased V2 dendritic fields; (D) five non-biased or mildly orientation-biased V2 dendritic fields; (E) four broadly morphologically direction-biased MT dendritic fields; (F) four non-biased MT dendritic fields; (G) four broadly direction-biased LIPv dendritic fields; (H) three non-biased and one morphologically complex LIPv dendritic fields. See also legend to Figure 11 for details. Cerebral Cortex Jul/Aug 1997, V 7 N 5 445 Table 6 Percentages of neurons with different categories of dendritic biases in visual areas* Visual area V1 V2 MT LIPv 7a Type of dendritic tree Morphologically Morphologically orientation-biased direction-biased Morphologically complex Nonbiased 45 5 15 10 10 – – – 15 15 15 20 50 35 20 40 75 35 40 55 *n = 20 for each area. regions at the origin of the occipitotemporal pathway, including V1 and the thick cytochrome oxidase-rich stripes of V2. Whereas there is general agreement that MT occupies a position intermediate in the proposed anatomical hierarchy, between V2 and posterior parietal areas LIPv and 7a, there is still controversy regarding the relative level of the latter two areas: some authors regard LIPv and area 7a as occupying the same hierarchical level (Boussaoud et al., 1990), whereas others suggest that area 7a is at a higher level than LIPv (Andersen et al., 1990; Felleman and Van Essen, 1991). Moreover, while V1, V2, MT and, to a lesser extent, LIPv are well-defined areas, it is unclear whether the subdivision of area 7a within the superior temporal sulcus is part of the same functional area as the subdivision occupying the caudal inferior parietal lobule, which has been most intensively studied in terms of connections (Andersen et al., 1990; Boussaoud et al., 1990). These caveats should be kept in mind when, in the following sections, we try to establish whether or not there are correlations between dendritic morphology and the proposed anatomical hierarchy. Neuronal Types Forming the V1 Projection to MT Various cell types, typically in layer III, have been demonstrated to form ‘feedforward’ projections between visual areas. These are typically classified within two main groups: pyramidal neurons and spiny multipolar (stellate) neurons (Tigges et al., 1981; Diamond et al., 1985; Shipp and Zeki, 1989a; Vogt Weisenhorn et al., 1995). We were particularly concerned with the cells forming the projection from V1 to MT, which, as indicated by our data, form a mixed population with a predominance of pyramidal neurons. Previous reports differ in their conclusions on this subject. This discrepancy may, in part, be attributed to species differences. For example, in the marmoset monkey all MT-projecting neurons in V1 are reported to be pyramidal (Vogt Weisenhorn et al., 1995), while in the squirrel monkey and the macaque multipolar neurons have been reported to form a substantial part of this projection (Tigges et al., 1981; Shipp and Zeki, 1989a). The relative proportions of pyramidal and multipolar neurons in different species do not bear any obvious relationship with either habits or phylogeny: in both the macaque (a diurnal Old World monkey) and the squirrel monkey (a diurnal New World monkey) many stellate cells are reported to project to MT, but the same is not true in the marmoset, another diurnal New World monkey. This variability suggests a developmental process regulating dendritic morphology, rather than a clear-cut separation between two categories. In fact, we obser ved many modified pyramidal neurons among the population of V1 cells that project to MT, some of which resembled multipolar cells in that their basal dendrites issued in all directions from the soma. Other authors have proposed that spiny multipolar cells represent an extreme 446 Supragranular Pyramidal Cells in Visual Cortex • Elston et al. Figure 13. Spine density as a function of distance from the cell body along basal dendrites of pyramidal neurons in five visual areas. The illustrated means and standard deviations are based on examination of 20 randomly selected basal dendrites from neurons in each area. Spine density was calculated per 10 µm increment of dendrite, and the arrowheads define the five consecutive segments that yielded the highest cumulative counts. Table 7 One-way ANOVA: peak spine density in different visual areas Source Sum of squares DF Mean squares F-ratio Probability Between areas Within areas Total 755.69 2407.61 3163.30 4 475 479 188.92 5.07 37.27 P < 0.0001 One-tailed between-groups comparison: Dunnett’s t procedure V2 MT LIPv 7a V1 V2 MT LIPv 5.11** 6.85** 5.80** 12.09** 1.69 0.79 7.24** 0.85 5.71** 6.36** ** P < 0.01. Table 8 Statistical comparison between cell body cross-sectional area Kruskal–Wallis test (DF = 4, n = 177, H = 114.32, P < 0.0001) Visual area Mean rank V1 V2 MT LIPv 7a 60.25 30 100.77 139.59 113.31 Mann–Whitney U-tests Comparison U′ z V1 versus V2 V1 versus MT V1 versus LIPv V1 versus 7a V2 versus MT V2 versus LIPv V2 versus 7a MT versus LIPv MT versus 7a LIPv versus 7a 913 648 915 843 1268 1726 1640 1019 732 1126 –4.94* –4.68* –6.03* –5.31* –6.50* –7.58* –7.14* –4.63* –1.51 –3.94* * P < 0.001. case of modified pyramidal neurons, involving the loss or reduction of the apical dendrite (Lund, 1984; Nieuwenhuys, 1994; Vogt-Weisenhorn et al., 1995). Methodological factors may also complicate the interpretation of the results of these different studies. For example, both Vogt Weisenhorn and colleagues (1995) and the present study, in which cellular morphologies were established by intracellular injection of retrogradely labelled neurons, concluded that the bulk of the projection from V1 to MT is formed by pyramidal neurons, whereas studies in which neuronal classification was based on retrograde labelling alone concluded that spiny multipolar neurons predominate (Tigges et al., 1981; Shipp and Zeki, 1989a). In addition, it is possible that the relative proportion of pyramidal and spiny multipolar neurons varies in different portions of V1 (Shipp and Zeki, 1989a). Note, for example, that Vogt-Weisenhorn and collaborators (1995), who found that only pyramidal cells give rise to projections to MT, dealt primarily with injections in the peripheral representation, whereas Tigges and colleagues (1981), who found mainly Figure 14. Frequency histograms of somal cross-sectional area of layer III pyramidal neurons in five visual areas. Somal areas of neurons in V1 were larger than those in V2 but smaller than those of neurons in MT, LIPv and area 7a. Somal areas of neurons in MT and area 7a were similar, but significantly smaller than those of neurons in LIPv. Arrows indicate means. Cerebral Cortex Jul/Aug 1997, V 7 N 5 447 Table 9 Somal cross-sectional areas of layer III pyramidal neurons in different visual areas Visual area n Mean (µm2) SD SEM Minimum (µm2) Maximum (µm2) V1 V2 MT LIPv 7a 24 44 31 39 39 170 126 218 283 233 6.9 6.0 6.0 12.0 6.8 129 71 160 172 167 279 259 297 586 334 34 39 33 76 42 multipolar neurons, studied the connections of the central field representation. In spite of the variable morphology, the laminar location (just above the granular layer) of the MT-projecting neurons in V1 is remarkably constant across primates, including prosimians (Diamond et al., 1985). This constancy suggests that the unifying feature across species is the ability to sample extensively, both directly and indirectly, from magnocellular inputs which terminate primarily in layer IVa (Fitzpatrick et al., 1985; Florence and Casagrande, 1987; Freund et al., 1989; Lachica et al., 1993). As recently demonstrated, pyramidal neurons in sublamina IIIc are inf luenced by parvocellular inputs onto their apical dendrites (Sawatari and Callaway, 1996); thus, the variability in the proportion of pyramidal cells may have functional consequences related to the amount of parvocellular input that reaches MT. In all retrogradely labelled pyramidal neurons in V1 in which we were able to visualize part of the axonal tree, there were horizontal radiations that projected laterally from the main axonal segment. At present it is unclear whether the horizontal axonal radiations of MT-projecting pyramidal neurons also contribute to clustered intrinsic connections in V1 (Rockland and Lund, 1983; Livingstone and Hubel, 1984; Amir et al., 1993; Lund et al., 1993). Intracellular injection studies in cat visual cortex (area 18) have revealed that intrinsically projecting pyramidal neurons have significantly smaller basal dendritic field widths than those that form extrinsic projections (Matsubara et al., 1996). Further experiments need to be performed to establish if this is also the case in the primate. Variation of Basal Dendritic Fields Between Areas, and Possible Functional Correlates Previous studies have demonstrated that anatomical parameters of intrinsic connections, such as the cross-sectional area and centre-to-centre spacing of intrinsic patches and the lateral extent of connectivity, increase from caudal to rostral visual areas (Yoshioka et al., 1992; Amir et al., 1993; Lund et al., 1993). Furthermore, Lund and colleagues (1993) have suggested that changes in cross-sectional area of intrinsic axonal patches are paralleled by changes in basal dendritic territories of supragranular pyramidal neurons. The present data confirm that, in some cases, basal dendritic fields of layer III pyramidal neurons increase with rostral progression along the proposed anatomical hierarchy of visual areas. However, they also demonstrate that a change of level in the hierarchy is not necessarily paralleled by changes in basal dendritic field area. Accepting that the size of basal dendritic fields of layer III pyramidal neurons is correlated with characteristics of intrinsic connectivity (Lund et al., 1993; Malach, 1994), we would expect to see a similar pattern of intrinsic axonal clusters in MT, LIPv and area 7a. It will certainly be worthwhile comparing the 448 Supragranular Pyramidal Cells in Visual Cortex • Elston et al. Figure 15. Dendritic field area as a function of somal cross-sectional area for pyramidal neurons in basal layer III in V1, V2, MT, LIPv and area 7a. Regression analysis revealed a significant correlation between these variables (r2 = 0.59). intrinsic connectivity of these areas in order to establish whether the proportional relationship between dendritic fields and intrinsic patches is maintained. Due to their morphology and location within the cortical column, layer III pyramidal neurons in extrastriate areas have the potential to sample from different types of inputs. As well as the intrinsic axonal clusters, pyramidal neurons at the base of layer III are in a position to sample ‘feedforward input’ (Rockland and Pandya, 1979; Felleman and Van Essen, 1991), feedback projections (some of which terminate in layer III, e.g. Boussaoud et al., 1990) and callosal projections (Cusick et al., 1984; Kennedy et al., 1986; Beck and Kaas, 1994). At present, little is known about the relative size and possible overlap of the multiple sources of inputs to cells in the rostral visual areas. In terms of connectional diversity (see Malach, 1994) it is feasible that neurons in layer III of rostral visual areas sample intrinsic and extrinsic input in different proportions. If we assume that the patchy nature of intrinsic and extrinsic connections is common to all visual areas (e.g. Rockland and Pandya, 1979; Montero, 1980; Van Essen et al., 1981, 1986; Pandya and Seltzer, 1982; Livingstone and Hubel, 1984; Caminiti and Sbriccoli, 1985; Seltzer and Pandya, 1989; Krubitzer and Kaas, 1990; Yoshioka et al., 1992; Amir et al., 1993; Lund et al., 1993; Shipp and Zeki, 1995; Fujita and Fujita, 1996; Leichnetz and Gonzalo-Ruiz, 1996; Seltzer et al., 1996), extrinsic inputs may form terminal patches that are smaller than, equal to or larger than the intrinsic axonal clusters in a given visual area. Thus, simply by varying the geometry of axonal patches and dendritic fields, neurons in different areas may be able to attribute different weights to different inputs and perform different functions. Aside from a correlation with intrinsic axonal patch size, it is possible that the observed increase in basal dendritic field areas of layer III pyramidal neurons between V1, V2 and MT, and the relative constancy beyond MT, are consequences of the fact that the present analysis was based on comparison of randomly sampled neurons in different areas, instead of specifically focusing on cells with similar projection patterns (intrinsic, extrinsic or interhemispheric). Current evidence suggests that high-order visual areas are generally connected to more cortical areas than are lower-order visual areas. For example, while layer III cells in V1 only form three main ‘feedforward’ cortical projections (Zeki, 1978; Weller and Kaas, 1983; Van Essen et al., 1986; Krubitzer and Kaas, 1993), with minor or variable pathways to a few other targets (Felleman and Van Essen, 1991; Rockland and Van Hoesen, 1994), cells in layer III of areas such as MT and area 7a may project to 10–20 other areas (Ungerleider and Desimone, 1986; Andersen et al., 1990; Felleman and Van Essen, 1991). Therefore it is possible that the proportion of neurons in layer III that form extrinsic projections is greater in higher-order areas than in V1 or V2. Given that interareal projection neurons tend to have larger dendritic trees than intrinsic projection neurons, as reported in cats (Matsubara et al., 1996), higher-order areas may have a greater percentage of large layer III pyramidal neurons. However, this explanation does not seem entirely satisafctory, as in areas MT and LIP the smallest neurons are bigger than the largest neurons in V1. Secondly, callosally projecting pyramidal neurons in layer III, which are on average larger than intrahemispheric projecting pyramidal neurons (Matsubara et al., 1996), are widespread in rostral visual areas such as MT, MST, LIP and area 7a. In contrast, in V1 and, to a lesser extent, V2 these cells are concentrated in the vicinity of the representation of the vertical meridian and fovea (Van Essen and Zeki, 1978; Newsome and Allman, 1980; Van Essen et al., 1982; Cusick et al., 1984; Kennedy et al., 1986; Olavarria and Abel, 1996). Thus, the greater number of inter- and intrahemispheric connections in rostral visual areas may, in part, be responsible for the overall trend towards large basal dendritic territories. (e.g. Leventhal and Schall, 1983), recent studies in cortex have emphasized the role of biased intrinsic axonal projections (e.g. Lund et al., 1995; Sincich and Blasdel, 1995; Fitzpatrick, 1996). We find it striking that a large proportion of cells with narrow morphologically orientation- and direction-biased dendritic fields were found in V1, where this property is first generated in the monkey cortex, but not in the other areas. These obser vations suggest the possibility that, in addition to the excitatory and inhibitory effects of intrinsic lateral connections (Vidyasagar et al., 1996), the orientation selectivity of layer III cells may be sharpened by sampling inputs from underlying layer IV along given axes of the visuotopic map. For dendritic field biases to be of any value in contributing to orientation selectivity, it is necessary that the given cortical area has a well-defined visuotopic map. This is certainly the case in V1, where cells in layer IV which provide inputs to layer IIIc form a fine-grained map of the visual field (Blasdel and Fitzpatrick, 1984), but not in the other areas studied (Rosa, 1997). In extrastriate areas the observed dendritic field biases may be more related to functional compartmentalization (cf. Katz et al., 1989; Hübener and Bolz, 1992; Kossel et al., 1995). For example, the cortical projections to these areas often terminate in a patchy pattern (Livingstone and Hubel, 1984, 1987; Ungerleider and Desimone, 1986; Cavada and Goldman-Rakic, 1989; Andersen et al., 1990; Boussaoud et al., 1990; Leichnetz and Gonzalo-Ruiz, 1996). Even though a cell body in these areas may be located relative to patches of axonal projection in a position to sample from different sources of input, its directional dendritic field could bias its responses in such a way as to ref lect mainly one of the inputs. Dendritic Field Complexity and Directional Biases Our results suggest that neurons in layer III of V1 and V2 differ from those in higher-order areas not only in that they have smaller basal dendritic field areas but also in the relative complexity of these fields. Even in the region of cortex 50–75 µm from the cell body, well within the lateral extent of the dendritic fields of V1 and V2 cells, there are relatively more dendritic branches per unit area in MT, LIPv and area 7a than in V1 and V2. The fact that maximal complexity was observed among neurons in LIPv, rather than in area 7a, suggests that there is no simple correlation between dendritic field branching complexity and position in the proposed hierarchy (although, as mentioned above, the level of the subdivision of area 7a that we studied is still uncertain). The fact that neurons in LIPv had, on average, a larger cell body than that of neurons in MT and area 7a (which were similar) may be related to the greater complexity of the dendritic field in the former area. The overall trend towards pyramidal neurons with more complex basal dendritic projections in areas MT, LIPv and area 7a may partially ref lect the relatively high proportion of neurons forming contralateral projections in these areas. For example, in area 18 of the cat, neurons that form callosal projections have more complex dendritic fields than those that form intrinsic or ipsilateral interareal projections (Matsubara et al., 1996). Our results suggest that there are significant orientation biases in dendritic fields of most neurons in layer IIIc of V1, that most neurons within the thick bands in V2 display some degree of dendritic field directional bias, and that these biases are less marked in neurons in higher-order areas (MT, LIPv and area 7a). While some models of generation of orientation selectivity in the nervous system suggest that dendritic field asymmetries may in part be responsible for the generation of this response property Spine Densities Our results indicate the existence of a significant variability in spine density along the basal dendrites of layer III pyramidal cells in all visual areas tested. These data are in agreement with previously published studies in the visual cortex of the rat (Larkman, 1991). Spine density of the basal dendrites of layer III pyramidal cells also differed markedly across areas (Fig. 13). We have shown significant differences in peak spine density between different visual areas of single animals, and between areas in animals of similar age. For example, neurons sampled from V1 and area 7a in a single 20-month-old animal (RM 13) were significantly different. Furthermore, the peak spine densities of V1 neurons in this animal were significantly different from those in areas MT and LIPv in another 20-month-old monkey (RM 14). Thus, any age-related causality for the different spine densities (Lund et al., 1977; Boothe et al., 1979; Courns et al., 1996) is unlikely. By combining the data from the Sholl and spine-density analyses we were able to calculate the average total number of basal dendritic spines for layer III pyramidal neurons in different visual areas. In spite of the approximate nature of the obtained figures, they serve as a relative measure for comparisons between areas, demonstrating a dramatic increase along the occipitoparietal pathway. It has been established that symmetrical synapses, which are thought to be excitatory, form the vast majority of contacts to dendritic spines (for review see Shepherd, 1996). We believe that the peak spine density, the spine distribution and the total number of spines, which are characteristic of different areas, may have functional significance. In the simplest case, one might consider two pyramidal cells with similar dendritic fields but different spine densities. In this case, it is reasonable to expect that the firing rate of the cell with greater spine density may be Cerebral Cortex Jul/Aug 1997, V 7 N 5 449 inf luenced by a greater diversity of excitatory input, particularly in view of the fact that only a small number of EPSPs may be needed to reach firing threshold in pyramidal neurons (Deuchars et al., 1994). This could allow the cell to sample from superimposed sources of differing origin; for example, juxtaposed feedforward input from multiple ‘low’ order areas, as well as intrinsic, feedback, thalamic and callosal projections. Furthermore, dendritic segments with greater spine densities may achieve greater summation of excitatory postsynaptic potentials, more easily depolarizing the target neuron even if only a subset of the dendritic tree is stimulated. However, the exact physiological effect of the varying spine density along dendrites is difficult to estimate, given a number of uncertainties regarding active versus passive centripetal propagation of electric activity in neocortical dendrites (for review see Johnston et al., 1996). Conclusions We have tested the hypothesis that there is a stepwise change in basal dendritic field morphology of supragranular pyramidal neurons from ‘lower’ to ‘higher’ stations of the proposed anatomical hierarchy which processes motion and spatial relationships. Our results yield some support to this view, in that the expected serial increase in basal dendritic field area is observed between areas V1, V2 and MT. However, the relationship breaks down at ‘higher’ stations such as LIPv and area 7a, as the basal dendritic field area of neurons in these areas is similar to that observed in MT. We have also observed other cytological correlates for the proposed hierarchy in the form of increases in the complexity of basal dendritic branching pattern and the density and total number of dendritic spines. These features may enable cells in the rostral areas to sample a greater diversity of inputs. A precise interpretation of the functional significance of these results will depend on a better understanding of the geometry of the pattern of axonal terminations from multiple origins onto single pyramidal cells. Acknowledgments We thank Dr David Vaney for allowing free access to his laboratory and, in particular, his intracellular injection apparatus. We are also greatly indebted to Dr David Pow for allowing us access to his superb antibody to Lucifer Yellow. 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