THE JOURNAL OF COMPARATIVE NEUROLOGY 311:445-462 (1991)

Architecture of Superior and Mesial Area 6 and the Adjacent Cingulate Cortex in the Macaque Monkey M. MATELLI, G. LUPPINO,

AND

G. RIZZOLATTI

Istituto di Fisiologia Umana, Universita di Parma, 43100 Parma, Italy

ABSTRACT The agranular frontal cortex is formed by several distinct functional areas. There is no agreement, however, on its cytoarchitectonic organization. The aim of this study was to redefine the cytoarchitectonic organization of superior and mesial area 6 and the adjacent cingulate cortex in the macaque monkey. A particular goal was to find out whether the so-called supplementary motor area (SMA) is cytoarchitectonically different from the rest of area 6 and whether it can be considered as a single, independent cytoarchitectonic area. The results showed that, rostral to F1 (area 4), four architectonic areas can be recognized in the superior (dorsal) and mesial area 6. Two of them are located on mesial cortical surface (F3 caudally and F6 rostrally) and two on superior cortical convexity (F2 caudally and F7 rostrally). The main cytoarchitectonic features of the five identified areas can be summarized as follows. F1: (1) giant pyramidal cells organized in multiple rows, (2) columnar pattern extending from the white matter to the superficial layers, (3)low cellular density in the lower part of layer 111. F3: (1)high cellular density in the lower part of layer 111, which fuses with a dense Va, (2) columnar pattern present only in the deepest layer, (3) occasional presence of giant pyramidal cells in layer Vb. F6: (1)prominent layer V, (2) absence of sublayer Vb, (3) homogeneous cell density in superficial layers. F2: (1)thin row of medium-size pyramids in the lowest part of layer 111, (2) columnar pattern extending to the superficial layers, (3) dense layer Va, (4) few, scattered giant pyramids in layer Vb.F7: (I) prominent layer V, (2) bipartite layer VI.Areas F1, F2, and F3, as defined cytoarchitectonically, coincided with the homonymous histochemical areas. The present data showed also that area 24 is formed by four subareas: 24a, b, c and d. Areas 24a and b occupy the ventral part of area 24, whereas its dorsal part is formed by area 24c, located rostrally, and area 24d, located caudally. The following features distinguish area 24d from area 24c: (1) larger pyramidal cells in layer V, (2) presence of medium-size pyramidal cells in the lower part of layer 111, (3) more prominent columnar pattern, (4) higher myelinization with the presence of an evident horizontal plexus. Mesial area 6 is usually considered as a single functional entity (SMA). Our findings show that this cortical region is formed by two distinct cytoarchitectonic areas, In the following article (Luppino et al. '91: J. Comp. Neurol 3 11:463-482) physiological evidence is presented that the SMA, as classically defined, corresponds to F3, whereas F6 is an independent functional area. Key words: premotor areas, area 4,area 24, cytoarchitectonics,primates

The agranular frontal cortex is classically subdivided into two large cytoarchitectonic areas: area 4 and area 6 (Brodmann, '09). This subdivision is mainly based on the distribution of giant pyramidal cells (Betz cells), which are abundant in area 4 but rare or absent in area 6. Although Brodmann ('09) considered area 6 as a single cytoarchitectonic area, differences among its various parts were described by several authors, notably by the Vogts ('19) and by von Bonin and Bailey ('47). According to the Vogts ('191, o 1991 WILEY-LISS, INC.

the dorsal aspect of monkey area 6 (superior area 6)and its mesial aspect (mesial area 6) are formed by two cytoarchitectonic areas: area 6aa, located caudally, and area 6ap, located rostrally (see Fig. 1).Von Bonin and Bailey ('471, although critical of the minute subdivisions of the Vogts, Accepted May 31,1991. Address reprint requests to G. Rizzolatti, Istituto di Fisiologia Umana, Universita di Parma, via Gramsci 14,43100Parma, Italy.

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von B o n i n H B a i l e y 1 9 4 7

Vogt

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Fig, 1. Parcellations of the agranular frontal cortex of the macaque monkey, according to 4 cytoarchitectonic maps. Dotted lines indicate the borders between the different areas. Hatched borders indicate transitions between areas marked by different color tonality in the map of von Bonin and Bailey ('47).

also concluded that area 6 is not homogeneous. They attributed most of mesial and superior area 6 to a single area, designated as area FB. A strip, however, between area FB and the prefrontal cortex, was segregated and described as area FC. In addition, von Bonin and Bailey ('47) found that inferior area 6 (the part of area 6 lying lateral to the spur of the arcuate sulcus) was structurally different from the other two area 6 sectors and subdivided it into two areas: FBA and FCBm. In the classical cytoarchitectonic literature, there is no mention of differences between mesial and superior area 6. This point of view held true even after the discovery that mesial area 6 contains an independent complete motor representation (supplementary motor area, SMA, Penfield and Welch, '51; Woolsey et al., '52). The possibility, however, that the SMA may have a structural counterpart has been recently suggested by two independent reports. Using cytochrome oxidase histochemistry, Matelli et al. ('85) described an area located on the mesial cortical surface,

which showed histochemical features different from all other sectors of area 6 (Fig. 2). This mesial area-called F3-extended caudorostrally for about 8-10 mm. F3 started just in front of area 4 (F1 in the terminology of Matelli et al., '85) and ended, approximately, at the same anteroposterior level of area 6aa of the Vogts. A mesial area, with its own specific characteristics, was described also by Barbas and Pandya ('871, using cell and fiber staining methods (Fig. 1).They called this area MI1 and considered it as the histological counterpart of the supplementary motor area. Surprisingly enough, MI1 of Barbas and Pandya ('87) did not border with area 4.MI1 was found to be separated from area 4 by a cortical strip, roughly parallel to area 4,which extended from the cingulate sulcus till the spur of the arcuate sulcus. This cortical strip was designated as dorsocaudal area 6 (6DC). The purpose of this study is to re-examine the cytoarchitectonic organization of superior and mesial area 6. Specifically, three points are examined. The first concerns the

CYTOARCHITECTURE OF SUPERIOR AND MESIAL AREA 6

Fig. 2. Parcellation of the agranular frontal cortex of the macaque monkey, according to the histochemical study of Matelli et al. ('85).

validity of a separation between mesial and superior area 6. As described above, previous cytoarchitectonic studies have either failed to differentiate these two sectors (Vogt and Vogt, '19; von Bonin and Bailey, '47) or described differences between mesial and superior area 6 only in their rostral parts (Barbas and Pandya, '87). The second point concerns the validity of parcellation of mesial area 6 into two areas, as originally suggested by the Vogts ('19). This point needs clarification since some authors, as, for example, von Bonin and Bailey ('471, attributed virtually the whole mesial area 6 to area FB, whereas others, such as Barbas and Pandya ('87), recognized a small caudal area (6DC) but assigned most of mesial area 6 to a rostral area (MII). The problem of the parcellation of mesial area 6 is particularly important, since it bears directly on the issue of the anatomical basis of the SMA. The third point involves the delimitation of the border of area 6 with the cingulate cortex. There is remarkable disagreement on this point among various investigators. Vogt et al. ('87) located the borders between area 6 and the cingulate cortex on the ventral bank of the cingulate gyrus, in close proximity to the fundus of the cingulate sulcus. Matelli et al. ('85) set the limits of area F3 much more dorsally, within the dorsal bank of the cingulate sulcus. According to von Bonin and Bailey ('47),both banks of the cingulate gyrus belong to an independent area (area FDL), which, basically, represents a caudal extension of the granular area FD. The border between area 6 and the cingulate cortex assumes particular interest because recent data showed that a contingent of corticospinal fibers originates from the cortex around the cingulate sulcus (Hutchins et al., '88). The present experiments showed that there are clear cytoarchitectonic differences between mesial and superior area 6 and that both these area 6 sectors are constituted of a caudal and a rostral part. The validity of this parcellation and, in particular, that of mesial area 6 into two areas was confirmed by physiological experiments, which are reported in the accompanying article (Luppino et al., '91).

METHODS The experiments were carried out on 6 macaque monkeys (5 Macaca fascicularis and 1 Macaca nemestrina). Each monkey was anesthetized with ketamine hydrochloride (15

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mg/Kg intramuscular) followed by an i. v. injection of Nembutal. The monkey was then perfused through the heart, first with 0.9% saline and then with 10% buffered formalin (pH = 7.4). The skull was opened and the dura incised. Photographs of the brain were taken to record the sulcal pattern. The brain was then blocked coronally on a stereotaxic frame, removed from the skull, and embedded in parlodion. The brains of 4 monkeys (seven hemispheres) were cut coronally, one brain (one hemisphere) was cut parasagittally, and one (one hemisphere) was cut horizontally from the dorsal cortical surface to the dorsal bank of the cingulate sulcus and then parasagittally along a plane perpendicular to the cingulate sulcus. All brains were cut at 40 km. Every fifth section was stained with the Nissl method and the adjacent section with the Woelke-Heidenhain method. Additional data were collected from 3 monkey brains cut frozen and stained for Nissl method. One series of sections was also processed for cytochrome oxidase histochemistry according to a protocol similar to that described by WongRiley ('79).The cytoarchitecture of the cortical areas of the brains cut frozen was compared with that of brains embedded in parlodion. No significant differences were found between the differently treated brains, The borders between the various identified cytoarchitectonic areas were plotted on individual section drawings and then reported on a brain view in which the mesial wall of the hemisphere was unfolded by using a procedure similar to that used by Woolsey et al. ('52). The nomenclature used in this study derives from and extends that adopted in a previous histochemical parcellation study (Matelli et al., '85). In analogy with von Economo ('29) and with von Bonin and Bailey ('47), the various fields identified in the agranular frontal cortex are referred to with the letter F (Frontal). In order to avoid confusion with previous classifications, the different areas are indicated with Arabic numbers. The precentral cortex is referred to as F1; the others have progressively higher numbers. A similar type of classification has been successfully adopted in the visual V2, etc.) (Talbot and Marshall, '41). cortex W1, The horizontal and parasagittal planes of section are optimal for comparing the laminar thickness of the different cytoarchitectonic areas located on the mesial and superior sectors of agranular frontal cortex, respectively. Therefore, cortical thickness and thickness of individual laminae were measured in the 2 brains cut with these planes of section. All measurements were made on those parts of each cortical area that optimally displayed the cytoarchitectonic features proper of that area. Cortical and laminar thickness values represent means of 50 measurements made in 5 different sections. Furthermore, in 3 different brains cut coronally, cortical thickness and thickness of layer I were also assessed in the identified agranular areas (50 measurements for each area). Finally, cortical thickness of layer V of the cingulate cortex were measured in one brain cut coronally (50 measurements for each area). Data from coronal sections were analyzed with a n ANOVA and subsequent pairwise comparisons, by using the Newman-Keuls method.

RESULTS Cytoarchitectonic organization of mesial agranular frontal cortex Figure 3 shows a low-power view of the cytoarchitecture of the mesial frontal cortex. The cortex was cut horizontally

Fig. 3. Low-power photomicrographs of two (A, B), Nissl-stained, horizontal sections, showing the cytoarchitedonic subdivisions of mesial agranular frontal cortex (Fl, F3, and F6). Left side is caudal. The level at which the sections were taken is shown in Figure 4a. Small arrows indicate the borders between cytoarchitectonicareas. PA and AS indicatethe level, on the

dorsolateral surface, of the genu of the arcuate sulcus and of the rostral end of the superior arcuate sulcus, respectively. C = central sulcus; PA = genu of the arcuate sulcus; AS = rostral end of the superior arcuate sulcus. Calibration bar = 1mm (A,B).

CYTOARCHITECTURE OF SUPERIOR AND MESIAL AREA 6

Monkey

1R

"(

W d

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Monkey 2 L

C

Monkey

3R

Fig. 4. Drawings of the 3 hemispheres from which selected sections were taken to show the cytoarchitecture of superior and mesial agranular frontal cortex. The drawings show the plan of cutting and the level (dashed lines) at which the selected sections were taken. (a)Mesial view. Horizontal cutting. Photomicrographs of sections A and B are shown in Figures 3,5,6, and 10. (b)Dorsolateralview. Parasagittalcutting. Photomicrographs of sections A, B, and C are shown in Figures 7,8,9,10, and 12. (c)Dorsal view. Coronal cutting. Photomicrographs of sections A, B, and C are shown in Figure 11.For others conventions, see Figure 3.

Fig. 5 (right). Photomicrographs of F1 (A), F3 (B), and F6 (C). Calibration bar = 500 km (A-C).

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F1

F3

F6

Fig. 6. High-power photomicrographs of F1, F3, and F6. The figure allows a direct comparison of lower part of layer I11 and layer V in the various areas. For details see text. calibration bar = 100 pm (FI,F3,F6).

at the levels shown in Figure 4a. Three agranular areas with different cytoarchitectonic organization can be recognized. They are referred to as F1 (area 41,F3, and F6. F1 is the caudalmost agranular frontal area. Absence of layer IV, poor lamination, and presence of giant pyramidal cells arranged in multiple rows characterize this area. In F1, unlike in the postcentral cortex, the upper part of layer V (Val is pale and poor in well-stained cells. Layer Vb is similar to Va in terms of cellular density but contains the giant pyramidal cells. Between F1 and the typical postcentral granular cortex, there is a transitional area (area 3a) characterized by an incipient granular layer N,a rich layer Va, and some giant pyramidal cells in layer Vb. The area next to F1 in the rostral direction is F3. Like F1, area F3 is poorly laminated. One of its most important distinguishing features is the increase in cellular density in the lower part of layer I11 and in layer Va. Layer Vb is pale as in F l . Giant pyramidal cells are absent in most of F3. They can be observed, however, in its caudal part, where, unlike in F1, they are arranged in a single row. Layer VI is darker than Vb. The third agranular area is F6. It has a dark, very evident layer V, well demarcated from the less dense layers I11 and VI. LayerVb, which is present in both F1 and F3, is lacking. F6 is the most rostral agranular frontal area. It borders a cortex in which an incipient layer IV becomes recognizable. A higher power view of the cytoarchitecture of the agranular frontal cortex is shown in Figure 5. At this magnification F1 appears to be formed by a series of vertical columns of cells parallel one t o another (Fig. 5A). This radial pattern can be recognized from layer VI until the upper part of layer 111.Layer I is thin. The border between layers I1 and I11 is difficult to delineate. The upper part of

layer I11 is dense and contains many small, darkly stained pyramids, whereas its lower part is pale. The cells in lower I11 are larger than in layer I1 and in upper I11 and tend to increase with the layer depth. The transition between layers I11 and Va is characterized by a slight increase of cellular density and by the disappearance of the mediumsize pyramids of lower layer 111. Two populations of cells are present in layer Vb: outstanding giant pyramidal cells and smaller intermingled pyramids. Giant pyramidal cells tend to cluster together and to be organized in multiple rows. Smaller pyramids have an overall density lesser than that of layer Va. The transition from layer Vb to layer VI is characterized by an abrupt change in cell type (fusiform and polymorph) and an increase in cell density (see also Fig. 6, Fl). A radial organization is recognizable also in F3. The vertical columns, however, are shorter in F3 than in F1 and stop abruptly in layer Va. The limitation of the radial pattern to the deep layers is accompanied by strong increase in cell density in layer Va and in the lower part of layer I11 (Fig. 5B). Layers I is thicker in F3 than in Fl(160 % 10 ym vs. 103 & 9 pm). Layer I11 is well developed and, unlike in F l , its lowest part is the densest. This part merges with

Fig. 7. Low-power photomicrographs of three (A, B, C ) , Nisslstained, parasagittal sections, showing the cytoarchitectonic subdivisions of superior frontal agranular cortex (F1,F2, and F7). Right side is caudal. The level at which the sections were taken is shown in Figure 4b. F2 in the medialmost section (A) shows a tendency of layer 111 to merge with layer Va. The fusion of lower layer I11 with layer Va is one of the basic characteristics of the adjacent F3. Conventions as in Figure 3. Calibration bar = 1mm (A-C).

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layer Va, which is also very rich in cells. The change in the organization of layers I11 and V starts in a part of cortex where giant pyramidal cells are still present. A comparison between the organization of lower layer I11 and layer Va in F1 and F3 is shown at a high magnification in Figure 6. The decrease in thickness of layer Vb, illustrated in Figure 6, was a constant feature of F3 (145 26 pm vs. 232 & 35 pm). Area F6 is rich in cells, but its cellular density is lesser than in F3 (Fig. 5C). This decrease in cellularity is particularly evident in the superficial layers. Layer I is thick (205 .t 9 Fm), whereas layer I1 is poorly developed as in the other agranular frontal areas. Layer I11 is fairly thick and uniform. It is mostly formed by small pyramids. Its border with layer V is sharp. Layer V is darkly stained and contains pyramids larger than those of layer I11 (Fig. 6).Although its extension in depth is irregular, its border with layer VI is easy t o delineate. Unlike in the other areas, layer V cannot be subdivided into two layers. Its thickness is virtually half of that of F1 layer V (248 27 pm vs. 455 ? 44 pm);

*

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Cytoarchitectonic organization of superior agranular frontal cortex Figure 7 shows a low-power view of the superior frontal cortex. The photomicrographs were taken at 3 different parasagittal levels (Fig. 4b). Three agranular areas with distinct cytoarchitectonic characteristics can be recognized: F1, F2, and F7. F1 borders with the granular parietal areas and extends rostrally for about 7 mm. Its cytoarchitectonic characteristics are indistinguishable from those of its mesial part. Rostral to F1 is area F2. Like F1, F2 is poorly laminated, but, unlike F1, it contains only few, scattered giant pyramidal cells. Layer I11 is characterized by a thin row of medium-size pyramids in its lowest part. Layer V is slightly denser and darker than in F1 (see also Fig. 9). These changes in the aspect of layers I11 and V are not so striking as those in F3 and, therefore, the border between F1 and F2 is less distinct than that between F1 and F3. F7 is the third agranular area recognizable in the figure. This area differs from F2 for two main characteristics: the presence of a prominent layer V and the subdivisionof layer VI into two sublayers. VIa is pale, whereas VIb is dark and dense. The area rostra1 to F7 shows an incipient layer IV and its laminar organization is more distinct than that of the agranular frontal cortex. These characteristics distinguish it from the agranular frontal areas. A higher power view of areas F1, F2, and F7 is shown in Figure 8. Like F1, F2 presents a radial organization from the deep to the superficial layers. It differs, however, from F1 for the following features. Layer I is thicker (191 14 pm vs. 163 t 21 pm). Layer I11 is more homogeneous in cell density than the homologous layer in F1. Its distinctive feature is the presence of a row of well-stained, mediumsize pyramids at the border with layer V. Layer V can be subdivided into two sublayers (Fig. 9). Layer Va is denser and thicker (284 .t 31 pm vs. 212 5 39 pm) than the corresponding layer in F1. In contrast, layer Vb is thinner (116 +- 23 pm vs. 193 & 32 pm) and is characterized by an almost complete absence of giant pyramidal cells. Unlike F1 and F2, F7 is an agranular area that shows a rather clear lamination (Fig. 8). The radial appearance

Fig. 8. Photomicrographs of F1 (A), F2 (B), and F7 (C). Calibration bar = 500 pm (A-C).

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F1

F2

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Fig. 9. High-power photomicrographs of F1, F2, and F7. The figure allows a direct comparison of lower part of layer I11 and layer V in the various areas. For details see text. Calibration bar = 100 km (Fl,F2,F7).

Fig. 10. High power photomicrographs of F6 (A), F7 (B), and areas 8b (C)and 9 (D) of Walker. The figure clearly shows that granules are present only in C and D. Calibration bar = 200 p.

observed in the other two areas is less evident. Layer I is thick (221 2 27 Fm). Layers I1 and 111fuse together. These two layers have a homogeneous aspect and contain mostly small pyramids. Medium-size pyramids are present, however, in the lowest part of layer 111. An evident, densely cellular layer V is one of the distinguishing characteristics of F7. This layer is thinner than in F1 and F2 (245 ? 20 pm vs. 405 ? 50 Fm and 400 2 31 pm, respectively) and similarly to F6 cannot be subdivided into two sublayers (see also Fig. 9). Within layer V the largest cells are usually located in its deepest part. Layer VI is formed by 2 sublayers, which contain mostly fusiform cells. These cells

are relatively sparse in VIa, whereas they are densely packed in VIb. In all the described areas, no granular cells were observed. These cells start to appear rostral to areas F6 and F7. This is illustrated in Figure 10, which shows a high power view of areas F6, F7, and of the rostrally located disgranular area 8b and granular area 9 of Walker. Note the presence in both these last areas of a distinguishable layer

Iv.

Figure 11 shows a series of coronal sections of the agranular frontal cortex, taken at different rostrocaudal levels (Fig. 4c). These sections allow a direct comparison of

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Fig. 11. Low-power photomicrographs of three Nissl-stained, coronal sections, showing the cytoarchitecture of mesial and superior frontal agranular cortex. The level at which the sections were taken is shown in Figure 4c. A. F6 and F7. B. F3 andF2. C. F1. Conventions as in Figure 3. Calibrationbar = 1mm (A-C).

the cytoarchitecture of adjacent areas located on mesial and dorsal cortical aspects. Note the well-developed layer V in F6 and the bipartition of layer VI in F7 (A). Note also the better lamination of F3 in comparison with F2 (B); C shows that no differences exist between the mesial and superior aspects of F1. This finding indicates that the cytoarchitectonic differences between superior and mesial areas rostral to F 1 are not due to their location on different cortical surfaces. Measurements of the thickness of the different cortical areas, made on coronal sections, confirmed the data obtained on horizontal and parasagittal sections. The values of the various areas were the following. Total thickness: F1= 2,466 2 85 pm, F2 = 2,162 2 34 km, F3 = 2,157 & 54 pm, F6 = 1,990 & 25 pm, F7 = 1,992 ? 31 pm. An analysis of variance and subsequent pairwise comparisons with the Newman-Keuls method showed that F l was different from F2 and F3, which were not different one from another. F1, F2, and F3 were significantly different from F6 and F7, which did not differ one from another. A measurement of layer I showed an opposite trend. This layer was thick rostrally and thin caudally. The values of layer I in the various areas were the following: F1 = 113 & 5 km, F2 = 154 ? 10 pm, F3 = 161 ? 9 pm, F6 = 189 & 17 Wm, F7 = 194 2 4 ym. An analysis of variance and subsequent pairwise comparisons with the Newman-Keuls method showed that F1 was different from F2 and F3, which were not different one from another. F1, F2, and F3 were significantly different from F6 and F7, which did not differ one from another.

Cytoarchitectonicorganization of the cingulate cortex (areas 23 and 24) Figure 12A shows a low-power view of the cytoarchitecture of areas 23 and 24. The cortex was cut parasagittally. The section crosses the superior frontal gyrus (areas F1 and F2), the dorsal bank of the cingulate sulcus (areas F1 and F3), and the ventral bank of the same sulcus (areas 23 and 24). Area 24 differs from area 23 for the following main aspects: (1)lack of layer IV, (2) presence of a thicker layer V (221 % 14 pm vs. 163 t 13 ym). This layer is paler and its pyramidal cells are less densely packed than in the homolo-

gous layer in area 23. A small transitional area can be recognized between areas 23 and 24. This area has the cytoarchitectonic characteristics of area 23 but with a less clear lamination and a thinner layer IV. Note that the posterior border of area 24 is caudal to that between F1 and F2 and is located approximately at the same anteroposterior level as that between F3 and F1. A complete reconstruction of the borders of the cingulate areas and their topographical relations with the agranular frontal cortex is presented below. Figure 12 (lower part) shows a higher magnification of layers 111, IV,and V of area 23 (Fig. 12B) and the corresponding part of area 24 (Fig. 12C). Granules are present only in area 23. A layer I11 rich of medium-size pyramids borders layer V in area 24. In agreement with previous studies (Rose, '27; von Economo, '29; Sarkissov et al., '55; Vogt et al., '87), area 24 was found to be formed by various subareas. These subareas, which have basically a ventrodorsal arrangement, are illustrated in Figure 13. The figure shows two coronal sections of the cingulate cortex taken at two different rostrocaudal levels. In the first section (A), three distinct subareas can be recognized: 24a, 24b, and 24c. Area 24a is poorly laminated with layers I1 and I11 fused together. Layer V is very dark and easily distinguishable from the superficial layers. In contrast, its border with the upper part of layer VI is difficult to set, given the richness in cells of this lamina. Area 24b shows a better lamination than area 24a. This is particularly evident in the deep layers were layer V is clearly separated by layer VI. The trend toward a well-developed lamination becomes more pronounced in area 24c, although also in this area layer I1 is difficult to separate from layer 111. Layer V is thin and paler than in 24a and b. Layer V contains medium-size pyramids. They are larger than those in the two previous areas. Layer VI shows a radial organization. It is formed by two sublayers (see also Fig. 14). The superficial layer is pale, whereas the deep one is almost as dark as layer V. Area 24c occupies the depth of ventral and dorsal banks of the cingulate sulcus as well as its fundus. Three subareas are evident also in the caudal section (B) of the figure. The first 2 subareas-24a and b-appear to be

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Fig. 12. A. Low-power photomicrograph of a Nissl-stained, parasagittal section, showing the cytoarchitecture of superior frontal gyrus (F1 and F2), dorsal bank of cingulate sulcus (F1and F3) and of the ventral bank of the cingulate sulcus (areas 23 and 24). Tr indicates the small transitional area located between area 23 and area 24. Conventions as

in Figure 3. Calibration bar = 1 mm. A higher magnification of the cytoarchitectonic features of areas 23 and 24 are shown in the lower part of the figure (B and C). B. Area 23. C. Area 24. Calibration bar = 100 km (B-C). For more details see text.

identical to those of section A. The third subarea, however, shows characteristics sufficient to distinguish it from subarea 24c. We refer to it as area 24d. The differences between areas 24c and 24d are particularly clear in parasagittal sections in which the 2 subareas are adjacent one to another (Fig. 14A). These differences can be summarized as follows. Layer V is thinner in area 24d than in area 24c (221 ? 14 km vs. 333 2 31 km) and its border with layer I11 better defined. The radial organization of deep layers is more pronounced in 24d than in 24c. In 24c the radial columns are observed only in layer VI, whereas in 24d they invade also layer V (see also Fig. 13).Finally, the cells in layers I11 (deep part), V and VI tend to be larger in 24d than in 24c. This point is clearly demonstrated in the right part of

Figure 14 (B and C) in which the two areas are shown at a higher magnification. The validity of a distinction between 24c and 24d was supported by the observation of the material stained for myelin. Figure lOAl shows a photomicrograph of areas 23, 24d and 24c stained with the Woelke-Heidenhain method. Area 24d, as well the transitional area between 24d and 23, appear to have a fiber content much richer then 24c. Radial fibers arrive to the lower part of layer I11 in area 24d, whereas they stop in depth of the cortex in 24c. A stripe of horizontally oriented fibers is evident in 24d, whereas it is absent in 24c. The different radial organization of the fibers is probably responsible for the different radial organization the two areas observed in Nissl-stained material.

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M. MATELLI ET AL. invade the dorsal cortical convexity for about 2 mm. (3) The ventral borders of F3 and F6 are located on the dorsal bank of the cingulate sulcus. About half of the bank belongs to these areas. (4) The ventral bank of the cingulate sulcus rostral to area 23 contains area 24d for most of its extent. This area borders dorsally with F3 and the caudal half of F6. In its rostral extension area 24d is narrow and, on the ventral bank of the cingulate sulcus, is progressively substituted by area 24c. ( 5 ) Area 24b runs beneath area 24c and 24d. It occupies the dorsal half of the cingulate gyrus, slightly invading the ventral bank of the cingulate sulcus.

DISCUSSION Organization of superior and mesial area 6 The present data show that there are clear cytoarchitectonic differences between mesial and superior area 6 and that both these sectors can be further subdivided into a rostral and a caudal part. Thus mesial and superior area 6 appear to be constituted of 4 distinct cytoarchitectonic areas: F2, F3, F6, and F7. The validity of rostrocaudal and mediolateral subdivisions of area 6 is discussed in separate sections. A final section is devoted to the connections of the four areas. Rostro-caudal organization o f superior and mesial area 6. The notion that the caudal part of superior and mesial area 6 differs from its rostral part is by no means new. Evidence in this sense was first provided by the Vogts ('19) in their classical study on cortex cytoarchitecture. These authors proposed to call the caudal sector of area 6, area 6aa, and the rostral sector, area 6ap. The validity of Vogt's parcellation was confirmed, at least for superior area 6, by subsequent stimulation experiments showing that motor responses could be easily evoked from 6aa, but not from 6ap (see Woolsey et al., '52). In spite of this finding, the distinction between caudal and rostral area 6 was ignored in most of recent literature on motor areas. Von Bonin and Bailey ('47) described an area FC, rostral to area FB (their equivalent of superior and mesial area 6), which, as FB, extended on mesial and superior aspects of the agranular frontal cortex. This area, however, for its location, extent and histological features (see von Bonin Fig. 13. Low-power photomicrographs of 2 Nissl-stained, coronal and Bailey, '471, appears to be a transitional area from sections, showing the ventro-dorsal arrangement of the cytoarchitecagranular frontal cortex to prefrontal cortex. FC cannot be tonic subdivisions of area 24. The 2 sections were taken at different considered, therefore, an equivalent of area 6ap. rostro-caudal levels. A. Rostra1 section, crossing F6. At this level, 3 Area 6 was subdivided into a caudal (6DC) and a rostral subdivisions of area 24 can be recognized: 24a, 24b, and 24c. B. Caudal (6DR) sector also by Barbas and Pandya ('87). These section, crossing F3. At this level, a further subdivision of area 24 can be recognized: 24d. Conventions as in Figure 3. Calibration bar = 1mm authors called 6DC that part of agranular cortex where the (A,3). Betz cells were present but scattered, and 6DR that part of agranular cortex where giant cells were absent altogether. Thus their 6DC corresponds to the caudalmost part of 6aa, whereas their 6DR corresponds to the rest of 6aa plus 6ap. Areal distribution Although the subdivision of Barbas and Pandya ('87) has its The location of frontal and cingulate areas in 4 hemi- internal logic, physiologicallyit is not convincing. Its weakspheres is shown in Figure 15c. Note that, in spite of some ness is evident if one examines the mesial surface where, variations in extent, the areal distribution is remarkably according to Barbas and Pandya ('87), an area (6DC) is constant. A similar areal distribution was found in the interposed between area 4 and the SMA. As shown in the accompanying article (Luppino et al., '91), this "area" is remaining monkeys of this study. Some points illustrated in the figure are worth noting. (1) nothing else but the leg representation of the SMA. In contrast to the above mentioned reports, our data fit The mesial part of F1 has a wedge-likeaspect with the apex directed toward the cingulate gyrus. The border between F1 well with the rostrocaudal parcellation of area 6 proposed and F3 is oblique and directed caudally and ventrally. ( 2 )F3 by the Vogts. They show that the boundary between the occupies approximately the caudal 213 of mesial area 6, two area 6 sectors lies slightly rostral to the junction of the extending sagitally for 8-10 mm; F6 occupies the rostral superior and inferior limbs of the arcuate sulcus. Table 1 third, extending sagittally for about 5 mm. Both areas shows a comparison between the present and previous

CYTOARCHITECTURE OF SUPERIOR AND MESIAL AREA 6

Fig. 14. A. Low-power photomicrograph of a Nissl-stained section, cut parasagitally perpendicular to the cingulate sulcus. Right is caudal. The cytoarchitectonic subdivisions of the dorsal part of area 24 (areas 24c and 24d) are presented. A l . Low-power photomicrograph of a Woelcke-Heidenhain stained section, adjacent to A, showing the dif-

457

ferent myeloarchitecture of areas 24c and 24d. The small arrows in A and A1 indicate the same blood vessel. Other conventions as in Figures 3 and 12. Calibration bar = 1 mm (A,Al). A higher magnification of areas 24c and 24d are shown in the right part of the figure (B and C). B. Area 24d. C . Area 24c. Calibration bar = 250 Frn (B,C).

parcellations of superior and mesial frontal agranular On the mesial aspect of the cortex, a strong increase in cortex (see also Figs. 1,2).The main features that character- cellularity can be observed at a certain rostral level in lower ize the two area 6 sectors are the following: (1)lamination is layer I11 and Va, which fuse together. A less strong but more developed in 6ap (F6-F7) than in 6aa (F3-F2); (2) the somehow similar modification can be seen also on the radial aspect of 6aa, observed from layer I11 to VI in F2 and cortical crown, where rostrally medium-size pyramids conin the deep layers in F3, is lacking in area 6ap, (3) layer V in centrate in the lowest part of layer 111. These changes in 6ap is formed by densely packed, small pyramids. This layer cellularity and organization of layer I11 were taken as a sign is much more distinguishable from layer I1 and I11 in 6ap of transition from F1 to caudal area 6 (F2 and F3). By using these criteria, the boundary between area F1 and caudal than in 6aa. A final point worth discussing is the caudal extension of area 6 (F2, F3) moves caudally in respect with that indiarea 6 and its boundary with area 4. Traditionally, area 4 cated in classical cytoarchitectonic maps. The physiological was defined as that sector of the agranular frontal cortex validity of this boundary is discussed in the accompanying which contains giant pyramidal cells (Betz cells) in its fifth article (Luppino et al., '91). It is interesting to note, layer. Conversely, area 6 was defined as that sector of the however, that (1)changes in layer 111 were considered of same cortex which does not contain them. The major fundamental importance by von Economo ("29) for distinproblem with these definitions is that, at least in the guishing FA (area 4) from FB (area 6) in humans and (2) monkey, Betz cells do not stop abruptly but rather become area 4, with the above mentioned cytoarchitectural criteria, rarer and rarer in a gradual way. Thus the decision of where coincides with that defined histochemically (Matelli et al., the giant pyramidal cells terminate, and, therefore, where '85). Mesio-lateral subdivision o f area 6. Areas 6aa and 6ap area 4 ends, becomes a rather arbitrary matter. In order to overcome the difficulties intrinsic to the are both formed by 2 parts, one located mostly on the mesial definition of area 4, we used two additional criteria to define cortical surface, the other on the dorsal surface. The F1, besides that of the presence of giant pyramidal cells. subdivision of 6aa into 2 areas, F2 and F3, was first The first criterion is the arrangement of these cells. In the proposed by Matelli et al., ('85) on the basis of a cytochrome caudal part of the agranular frontal cortex, giant pyramidal oxidase study of the agranular frontal cortex. They noted cells form an almost continuous multilayered row in layer that the various enzymatic bands, typical of motor cortices, V, whereas rostrally they are arranged, when present, in a have a more regular and neat organization on mesial cortex single line. We consider the agranular frontal cortex show- rostral to area 4,than on the dorsal convexity. The notion of a diversity between mesial and dorsal ing this last arrangement as part of area 6 and not of F1. The second criterion concerns the organization of layer 111. aspects of area 6 was confirmed by Barbas and Pandya

M. MATELLI ET AL.

458

C AS

PA

i

1

\

F2

F7

AS

4A

1

I

F7

F2

t C

Fgc

F3

F6

B

F1

.... . ..._..----

- .- ...

24 c

C

Skc

--. -._

---.....-

24d 23

D

E Monkey

5 R 5 rnrn

c--L-1 PA

AS

1

i F7

F2

f

AS

PA

i

t

C

F1

Figure 15

F3

F6

FQc

CYTOARCHITECTURE OF SUPERIOR AND MESIAL AREA 6

459

TABLE 1. Subdivisions of Superior and Mesial Frontal Agranular Cortex'

Matelli et al., '91

Vogt and Vogt, '19

Von Bonin and Bailey, '47

Barbas and Pandva. '87

Functional

~

Brodmann area 4 Superior

Rostrd Caudal

F1 F7 F2

Mesial

Rostral Caudal

F6 F3

Brodmann area 6

FA FB + FC FB

4 6DR 6DC + 6DR

Precentral motor cortex

FB FC FB

+

MI1 6DC + MI1

SMA? SMA

SEF PM

'Subdivisions of infenor area 6 (F4,F5) are not presented in this table

('87). These investigators found that in mesial area 6 the cells in layer I11 and V are more densely packed and densely stained than in superior area 6. The present data confirm these findings. Furthermore, they indicate that the extension of F3, as determined cytoarchitectonically, corresponds to that observed in histochemical material. In the accompanying article (Luppino et al., '911, it is argued that the SMA, defined as an electrically excitable area that possesses a complete independent motor representation, is co-extensive with F3. The present findings show also that a subdivision into a mesial (F6) and superior (F7) sector is valid for the rostral area 6 as well as for the caudal one. The main characteristics which differentiate the two rostral sectors are: (1)layer V is darker and more prominent in F6 than in F7, and (2) layer VI is subdivided into two sublaminae in F7 but not in F6. A different organization between mesial and dorsal cortical aspects has been observed also in the prefrontal cortex (Barbas and Pandya, '89). Following Sanides ('72), Barbas and Pandya ('89) interpreted this finding as a reflection of subsequent evolutionary waves leading from the hippocampal allocortex to the well-laminated isocortex. It is outside the scope of this study to discuss the validity of Sanides' theory. What is important, however, here is to stress that no difference in the cytoarchitectonic pattern was found between the mesial and dorsal aspect of F1 (area 4). This finding indicates that, regardless of their evolutionary origin, the differences between mesial and superior area 6 reflect a different anatomo-functional organization. They are not a mechanical consequence of the different curvature of the cortex on its dorsal and mesial surfaces. Connections o f superior and mesial area 6. The existence of 4 areas in superior and mesial area 6 raises the problem of their anatomical connections. In this section we review the thalamo-cortical and the frontal connections of the 4 areas in order to verify whether the proposed distinc-

tion is supported by a differential connectivity. With few exceptions, the reviewed data are taken from experiments in which areas were defined using criteria different from those proposed in the present study. This fact limits, considerably, the amount of unambiguous material. In many cases, however, terms such as rostral, caudal, dorsal, and medial specify, in a sufficiently clear way, which cytoarchitectonic area was studied. As far as the thalamo-cortical connections are concerned, several studies described the thalamic input to mesial area 6, often designated as the SMA. A finding that emerges from these studies is the differential thalamic input to rostral and caudal parts of this "area." The caudal part of it receives its main thalamic projections from nucleus ventralis lateralis, pars oralis (VLo, Olszewski, '52), with some additional contribution from nucleus ventralis lateralis, pars caudalis (VLc) (Schell and Strick, '84; Wiesendanger and Wiesendanger, '85). In contrast, the rostral part of mesial area 6 is mainly connected with area X of Olszewski and with the nucleus ventralis anterior, pars parvocellulark (VApc),with a lesser projection from VLc (Miyata and Sasaki, '84; Wiesendanger and Wiesendanger, '85). This differential thalamic input fits well with our conclusion that mesial area 6 is formed by two areas. A conclusion that becomes even stronger if one considers that VLo and VApc, although both recipient of basal ganglia output, are considered to belong to two different cortico-striate circuits (Alexander et al., '86). If this is accepted, one should conclude that F3 is an area receiving mostly a somatotopically organized input coming from the putamen (via VLo), whereas F6 is a "higher" order area connected with the caudate nucleus (via VApc). Finally, both areas receive a cerebellar input, although again from two different thalamic nuclei. In contrast to mesial area 6 , only few data are available on the thalamic input to superior area 6. In spite of this, a differential innervation of its rostral and caudal parts appears rather clearly from previous investigations (Kievit and Kuypers, '77; Miyata and Sasaki, '83; Darian-Smith et Fig. 15. (a)Mesial view of one hemisphere; dotted lines indicate the al., '90). The caudal part receives mostly from VLo andVLc, borders of the cytoarchitectonic areas; arrows indicate the level of the genu of the arcuate sulcus (PA) and the rostral end of the superior whereas the rostral sector receives from area X and VA arcuate sulcus (AS); C = central sulcus; Cg = cingulate sulcus. (b) complex. Of great interest is the differential thalamic Reconstruction procedure of the mesial cortical surface. (c) Distribu- projection to F6 and F7. F6 receives from the central part of tion of cytoarchitectonic areas in frontal and cingulate agranular area X and from VApc, whereas F7 receives from the cortex. Drawings of 4 hemispheres. The mesial surface of the hemimedialmost part of area X and from nucleus ventralis sphere is unfoldedto show the cortical surface buried into the cingulate sulcus. The 2 thick solid lines represent the reflection of superior anterior, pars magnocellularis (VAmc) (Miyata and Sasaki, '83; Miyata and Sasaki, '84; Wiesendanger and Wiesendansurface into the mesial surface and the corpus callosum, respectively. The dorsal thin solid line indicates the reflection of mesial cortex into ger, '85; Shook et al., '89). One difference between VApc the dorsal bank of the cingulate sulcus. The ventral thin solid line and VAmc is that the latter nucleus is primarily related to indicates the reflection of the ventral bank of the cingulate sulcus into oculomotor control. This distinction is congruent to the fact the cingulate gyrus. The dashed line indicates the fundus of the that F7 (or at least its medial part) is coextensive with the cingulate sulcus. Dotted lines mark the cytoarchitectonic borders. Fgc = Frontal granular cortex. Skc = Somatic koniocortex. Other supplementary eye field (Schlag and Schlag-Rey, '85, '87; conventions as in Figure 3. Huerta and Kaas, '90; Luppino et al., '91), whereas in F6

460 arm movements are mostly, although not exclusively, represented (Rizzolatti et al., '90; see also Mann et al., '88). Further evidence for a n independence of the 4 areas forming superior and mesial area 6 comes from a study from our laboratory in which the cortico-cortical connections within the frontal lobe were studied after tracer injection in F3, F6, and F7 (Luppino et al., '90). The results can be summarized as follows. F3 is the only area of the 3 injected that is connected with F1 (area 4). F3 and F6 are both connected with the other premotor areas, but their connection pattern is not identical. F3 is mostly linked with posterior areas (F2 and F4), whereas F6 is richly linked with the anteriorly located F5. Most interestingly, F6 is connected with the prefrontal cortex (area 46), whereas this link is absent in F3. Finally, F7 has a connection pattern markedly different from F3 and F6. The part of F7 in which tracer was injected was the medial one, near F6, i.e., that part from which eye movements could be evoked with electrical stimulation. After injection, labelled cells were found in area 8 and in prefrontal cortex. None of the motor or premotor areas controlling body movements was labelled. The distinction between areas which have connections with area 4 (F2 and F3) and areas which lack these connections (F6 and F7) (see in addition to the above described data, Matsumura and Kubota, '79; Muakkassa and Strick, '79; Godschalk et al., '84; Leichnetz, '86; Matelli et al., '86; Ghosh et al. '87) has an important counterpart in the organization of corticospinal efferents. There is a rich evidence, coming from degeneration studies (see Kuypers, '81) as well as from research with anterograde and retrograde tracers (Biber et al., '78; Murray and Coulter, '81; Toyoshima and Sakai, '82; Hutchins et al., '88; Keizer and Kuypers, '89; Nudo and Masterton, ,901, that corticospinal pathways originate from area 6aa (F2 and F3) but not from area 6ap (F7 and F6). Particularly interesting is a recent study by Keizer and Kuypers ('89) in which fluorescent tracers were injected in the spinal cord at C2 level and in the medial tegmentum of the medulla oblungata. As far as the superior and mesial area 6 are concerned, they found 3 cortical sectors characterized, respectively, by projections almost exclusively to the spinal cord, by projections to the spinal cord and to the brainstem, and by projections to the brainstem only. Keizer and Kuypers ('89) did not subdivide the cortex into cytoarchitectonic areas. However, a direct comparison of their zones and the cytoarchitectonic parcellation proposed in the present study show a surprisingly accurate match. F1 corresponds to the zone which projects to the spinal cord only, F2 and F3 correspond to the zone rich of double-labelled cells, F7 and F6 correspond to the zone which does not project to the spinal cord but has rich connections with the brainstem. Finally, a survey of the finding on the distribution of the cortico-descending pathways from F7 confirms the specific relation that this area has with eye movements organization. Evidence in this sense is provided by the experiments of Shook et al. ('88) and Huerta and Kaas ('90). Similarly, a different pattern of descending projections from area F7 on one side and F6 and F3 on the other has been demonstrated by Fries ('85).After injection of HRP in the deep layers of the superior colliculus, he found labelled cells in F7 and, more rostrally, in the frontal granular cortex. In contrast, no labeled cells were observed on the mesial aspect of the hemisphere, neither in F3 nor in F6.

M. MATELLI ET AL.

Organization of the agranular cingulate cortex Although the notion that the cingulate cortex may participate in the control of movements is by no means new (Smith, '45; Ward, '48; Kaada, '51; Showers, '59; Van Buren and Fedio, '761, until recently it received little attention from investigators in the motor field. Recent findings, however, on the connections of cingulate areas (Muakkassa and Strick, '79; Godschalk et al., '84; Leichnetz, '86; Morecraft and Van Hoesen, '88; Luppino et al., '90) as well as some occasional physiological data indicating that body movements can be occasionally elicited with intracortical stimulations of area 24 (see Macpherson et al., '82; Mitz and Wise, '87) re-opened the question of motor functions of the cingulate gyrus. In this new context, it was important to re-examine the cytoarchitecture of area 24 in order to provide a good starting point for further physiological examinations. In agreement with previous observations, the present findings showed that the cingulate cortex lying ventral to the agranular frontal cortex is formed by two clearly distinct areas: area 23 and 24. The first area has a welldeveloped layer IV, whereas the second area has an agranular structure. The border between the granular cingulate cortex (area 23 plus the disgranular transition) and 24 runs obliquely in a dorsoventral and caudorostral direction underneath F1 and the caudal part of F3. If one examines the location of the cingulate border with respect to the border between F1 and F2, it is clear that it runs posteriorly to that between these motor areas. This location is consistent with the results of most previous investigators (see Vogt et al.,'87). An exception is the study of Hutchins et al. ('88), who, surprisingly, extended area 23 for several millimeters in a rostral direction below F3. It is not clear, however, if this location was based on cytoarchitectonics or was derived, a posteriori, from the corticospinal connection pattern (see below). The dorsal border of area 24 is less easy to delimit than the caudal one. The difficulty is mostly due to the bending of the cortex around the cingulate sulcus and, as a consequence, to the necessity of comparing slabs of cortex oriented differently. It is likely that this difficulty led some investigators to put the boundary between area 24 and 6 in the correspondence to the sulcus depth (Barbas and Pandya, '87; Vogt ed al., '87). The present findings indicate that the internal third and, in some points, half of the dorsal bank of the cingulate sulcus belong to area 24. This location of the dorsal border was confirmed in myelin-stained material as well as in sections reacted for cytochrome oxidase (see also Matelli et al., '85). Area 24 is not homogeneous (Rose, '27; von Economo, '29; Sarkissov et al., '55). According to Vogt et al. ('87), in the monkey it is formed by three subareas: 24a, 24b, and 24c. These subareas run in a rostrocaudal direction and are, roughly, parallel to the corpus callosum. Area 24a, which is adjacent to the callosal sulcus, is the least differentiated, whereas the dorsalmost area, 24c, shows the best lamination. The present findings are in general agreement with the results of Vogt et al. ('87). They show, however, that area 24c is not homogeneous but constituted of 2 sectors: a caudal one, which we called area 24d, and a rostral one for which the original denomination (24c) was maintained. Area 24d is characterized by the presence of pyramidal cells larger than those observed in area 24c in layer I11 and

CYTOARCHITECTURE OF SUPERIOR AND MESIAL AREA 6 especially V. Furthermore area 24d has a well-evident radial vertical organization. To this organization, observed in Nissl-stained material, corresponds a richer vertical fiber plexus and a more prominent horizontal fiber distribution than in area 24c. In more general terms the cytoarchitectonic trend from 24c to 24d is somehow similar to that from area 6 to area 4. Large cells and a more marked radial appearance characterize the caudal agranular areas of both cingulate and frontal cortices. It is interesting to note that a paralimbic primitive gigantopyramidal field, with the characteristics of a “primordial” motor area, has been described also in man (Braak, ’76). This field is totally buried into the depth of cingulate sulcus and is located just rostrally to area 4, extending in a rostrocaudal dimension for about 18 mm. The architectonic features and the location of this field suggest that this primordial motor area could correspond to area 24d of the monkey. Cingulate neurons projecting to the spinal cord have been described both in area 23 and in area 24 by several authors (Biber et al., ’78; Murray and Coulter, ’81;Macpherson et al., ’82; Toyoshima and Sakai, ’82; Hutchins et al., ’88; Keizer and Kuypers, ’89).According to Hutchins et al. (’881, there are 2 forelimb and hindlimb representations: one rostral, located in area 24c of Vogt et al. (’871, and one caudal located in area 23. As mentioned above, Hutchins et al. (’88) extended rostrally area 23 for several millimeters below the supplementary motor area (F3). The parcellation of area 24 found in the present study suggests a different interpretation of their findings. A direct comparison of the labelled areas with our cytoarchitectonic maps indicates that there is one forelimb and hindlimb representation in area 23 and 2 representations in area 24. Of these, that located in area 24d contains both a forelimb field (located caudally) and a hindlimb field (located rostrally). The second representation, lying in area 24c, contains only a forelimb field. Consistent with this interpretation are the results of electrical stimulation of cingulate cortex presented in the accompanying article (Luppino et al., ’91). Those data indicate that area 24d is electrically excitable with relatively low currents and that in area 24d there is a representation of both the arm and the leg. A further arm representation was found in area 24c. Thus in area 24 there are 2 arm representations, one in each of the dorsal subareas, and certainly one leg representation (area 24d). Further experiments are necessary to find out whether also area 24c has a leg field.

ACKNOWLEDGMENTS The work was supported by EEC contract no SC1*0177-C and by grants from CNR and MPI to G.R.

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