The lateral premotor cortex plays a crucial role in visually guided limb movements. It is divided into two main regions, the dorsal (PMd) and ventral (PMv) areas, which are in turn subdivided into functionally and anatomically distinct rostral (PMd-r and PMv-r) and caudal (PMd-c and PMv-c) sub-regions. We analyzed the callosal inputs to these premotor subdivisions following 23 injections of retrograde tracers in eight macaque monkeys. In each monkey, 2–4 distinct tracers were injected in different areas allowing direct comparisons of callosal connectivity in the same brain.
Based on large injections covering the entire extent of the corresponding PM area, we found that each area is strongly connected with its counterpart in the opposite hemisphere. Callosal connectivity with the other premotor areas, the primary motor cortex, prefrontal cortex and somatosensory cortex varied from one area to another. The most extensive callosal inputs terminate in PMd-r and PMd-c, with PMd-r strongly connected with prefrontal cortex. Callosal inputs to PMv-c are more extensive than those to PMv-r, whose connections are restricted to its counterpart area. Quantitative analysis of labelled cells confirms these general findings, and allows an assessment of the relative strength of callosal inputs.
PMd-r and PMv-r receive their strongest callosal inputs from their respective counterpart areas, whereas PMd-c and PMv-c receive strong inputs from heterotopic areas as well (namely from PMd-r and PMv-r, respectively). Finally, PMd-r stands out as the lateral premotor area with the strongest inputs from the prefrontal cortex, and only the PMd-c and PMv-c receive weak callosal inputs from M1.
The motor cortex of macaques is divided into four main regions: the primary motor cortex (M1), the premotor cortex (PM), the supplementary motor area (SMA) and the cingulate motor area (CMA). These regions have been subdivided further into distinct areas on the basis of anatomical and functional criteria. In the PM region, the dorsal (PMd) and ventral (PMv) areas have been distinguished on anatomical, histochemical and neurophysiological ground [1-6]. More recently, PMd and PMv have been proposed to contain distinct functional areas along the rostro-caudal axis, referred to as PMd-r, PMd-c, PMv-r and PMv-c [5,7-9]. They correspond roughly to areas F7, F2, F5 and F4, respectively, in the nomenclature of Matelli and his co-workers [10-14]. Similarly, SMA has been subdivided into a rostral part (pre-SMA) and a caudal part (SMA-proper) , also referred to as F6 and F3, respectively [10,11,13,14,16]. Finally, three areas have been identified within the CMA on the basis of corticospinal projections : a rostral area (CMA-r) and two caudal areas, one dorsal (CMA-d) and one ventral (CMA-v). These multiple subdivisions are illustrated in Figure 1.
Figure 1. Premotor areas represented on a two-dimensional map of the cortex. On the left, surface view of the anterior part of the right hemisphere. The rectangle indicates the cortical region flattened and shown on the right. On the 2-D map, sulci are represented by shaded zones, the dashed lines indicate the fundus of the sluci. The premotor subdivisions are defined on the basis of SMI-32 staining (see text). Abbreviations: Ar, arcuate sulcus; Ce, central sulcus; CgG, cingulate gyrus; Ci, cingulate sulcus; CMA-d, r and v, dorsal rostral and ventral parts of the cingulate motor area, respectively; P = sulcus principalis; M1, primary motor cortex; PMd-c, r, caudal and rostral parts of the dorsal premotor cortex; PMv-c, r, caudal and rostral parts of the ventral premotor cortex; pre-SMA, rostral part of the SMA; SMA-proper, caudal part of the SMA.
The ipsilateral connections of these motor cortical areas with the other cortical areas have been extensively studied since many years, with renewed interest in recent years especially with respect to the posterior parietal cortex [1,7,10-12,18-60]. By contrast, callosal connections of most premotor areas have attracted less attention, despite their importance for understanding inter-hemispheric exchange of information necessary for coordinated actions of the two sides of the body . It is thus of interest to know how each premotor area connects with the opposite hemisphere in terms of topography and strength of the connections. Previous studies have described the callosal connectivity of M1 and SMA-proper [31,61,62]. They have shown that the hand area of M1 receives a minor callosal input from its counterpart in the other hemisphere, whereas the hand area in SMA-proper is more densely interconnected with the other hemisphere. More recently, Liu et al.  have contrasted the callosal connections of SMA-proper and pre-SMA and found that the two areas share common callosal inputs but the strength of the connections differs, with pre-SMA more heavily connected with the opposite hemisphere.
Callosal connectivity of the other premotor areas has been less investigated. Only one recent study  has described the callosal connections of the rostral and caudal dorsal premotor areas (PMd-r and PMd-c, corresponding to the areas F7 and F2, respectively), whereas those of ventral premotor and cingulate motor areas are still lacking. We performed an extensive multiple tracing investigation of callosal connections of the lateral premotor areas, with emphasis on the distinction between dorsal (PMd) and ventral (PMv) sectors as well as the comparison between their rostral and caudal divisions. We compared data obtained from two groups of animals. In the first group, large injections of 3–4 tracers were performed in each animal (n = 3) filling in most, if not the whole, extent of the PM sub-areas. In a second group of animals (n = 5), we performed smaller injections in the PM sub-areas for comparison with other studies. The first group of animals was used to describe a fairly exhaustive picture of the origin of the callosal projection to the four sub-areas of PM, including the issue of overlap/segregation of the different projections, whereas more precise topographic aspects are described based on the second group of animals.
The locations of the injection sites were confirmed on histological criteria. Figure 2 shows the reconstruction of each injection site on surface views of the brain hemisphere. Each monkey received 2–4 distinct tracers injected in different PM areas. As the figure shows, the injection sites varied in size and location within each PM sub-area and, sometimes, encroached on an adjacent area (see also Table I). A particular protocol was conducted in Mks 1–3 in order to obtain large injection sites covering most of the injected PM sub-area. Examples of such large injection sites are shown on photomicrographs (Fig. 4), following injections of BDA, DY, FB and CB. The injections were performed in such a way (usually at 2 depths along each penetration) to form a cylinder covering all cortical layers, from the surface down to the limit between the grey and the white matter.
Figure 2. Reconstruction of the injection sites on a lateral view of the left hemisphere of the 8 monkeys included in the present study.
Figure 3. Photomicrographs showing SMI-32 staining observed in Mk2 or Mk3 illustrating transition zones between the prefrontal cortex (Pfc) and PMv-r (panel A), between PMd-c and M1 (panel B), between PMv-r and ProM (panel C) and PMv-c and SomC (panel D). See list of abbreviations. Scale bar = 1 mm.
Table 1. List of tracers injected in PM in Monkeys Mk1 – Mk8, with indications on the total volume injected for each tracer, the number of penetrations and sites of infusions.
Figure 4. Photomicrographs of typical injection sites for BDA (left column) and for DY, FB and CB (from top to bottom in the right column). Scale bar = 1 mm.
In the following sections, we will describe the callosal labelling based on representative examples of the results obtained in MK1 and MK2 (Fig. 5 and 6). Individual variability is illustrated in figure 8 for 11 cases, and additional tables  and a figure  are presented as Supplementary material. In Figures 5 and 6, we have chosen to superimpose the labelling from 4 different tracers for comparison reasons.
Figure 5. Frontal sections of the right hemisphere of Mk1, arranged from rostral to caudal with increasing ID# (10 to 54), showing the distribution of retrogradely labelled neurons as a result of tracers injections in the opposite PMd-r (red dots), PMv-r (grey dots), PMd-c (blue dots) and PMv-c (green dots). The tracers used are indicated in the bottom right.
Figure 6. Frontal sections of the right hemisphere of Mk2, showing the distribution of retrogradely labelled neurons as a result of tracers injections in the opposite PMd-r (red dots), PMv-r (grey dots), PMd-c (blue dots) and PMv-c (green dots). Same conventions as in Figure 5.
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Injections in PMd-r
Five injections were made into PMd-r (Table I). Figures 5 and 6 (red dots) illustrate the distribution of retrogradely labelled cells in the hemisphere contralateral to the injection site following large injections into PMd-r of two monkeys. As the figures show, PMd-r receives it main callosal projections from the premotor areas anterior to the level of the genu of the arcuate sulcus, and from prefrontal cortex. Within this region, labelling was assigned to PMd-r, Pfc dorsal to the principal sulcus extending to the cingulate sulcus, pre-SMA and rostral cingulate cortex (CMA-r). In the cortex located laterally or caudally to the level of the genu of the arcuate sulcus, labelling was sparse or limited to small patches (Pfc ventral to the principal sulcus, PMv-r, PMd-c, CMA-v). This general pattern of transcallosal labelling was consistent with the data derived from a smaller injection of DY in PMd-r in Mk 6 (see Fig. 7, green patches), although the labelling was less extensive.
Figure 7. Distribution of labelling after 3 injections in Mk6 illustrated on a 2-D map of the frontal cortex. Same abbreviations and conventions as in Fig. 1.
Figure 8. A: quantitative data giving the percent distribution of callosal neurons observed in different cortical areas as a result of tracer injections made in PMd-r (top left plot), PMd-c (top right plot), PMv-r (bottom left plot) and PMv-c (bottom right plot). The percent values are given by different symbols for each of the three individual monkeys included in the quantitative analysis (Mk1, Mk2 and Mk3). For a given monkey, the sum of the percent values is 100%.
Injections in PMd-c
Seven injections were made in PMd-c in 7 animals (Table I). Figures 5 and 6 illustrate the distribution of labelling on coronal sections (blue dots), and figure 7 shows the data for monkey Mk6 on a 2-D map of the cortex. As after injections in PMd-r, injections in PMd-c yielded extensive labelling in the dorso-medial frontal cortex of the contralateral hemisphere. The main difference is that here, the labelling was relatively more caudal than following injections in PMd-r (compare red and blue dots). Analysis of the distribution of labelled cells in relation with areal borders shows that the strongest labelling was located in PMd-c, PMd-r and pre-SMA. Moderate or weak labelling was also found in the cingulate motor areas (CMA-r, CMA-v and CMA-d), SMA-proper and M1. The general pattern of labelling was the same in Mk3 where large injections were made, except that the labelling was more predominant in PMd-c than in PMd-r (see Fig. 8A and supplementary material). The results following a small injection in PMd-c are shown in Fig. 7, and they confirm the main observations made on the basis of large injection. The main difference is that the labelling was less extensive in rostral PMd-r following a small injection.
Injections in PMv-r
Six injections were made in PMv-r of 6 different animals (Table I). The key finding is that following these injections, most labelled cells in the contralateral hemisphere were found in the cortex located just behind the inferior arcuate sulcus, anterior to the level of the genu (sections 14–22 in Fig. 5 and 6, grey dots), which corresponds to the counterpart area PMv-r. As one moves anteriorly or posteriorly, the dense labelling in PMv-r moves ventrally, forming a long stripe within the bank of the lateral sulcus (Fig. 5 and 6; see also supplementary figure). At its caudal aspect, this labelling is probably in area S2. Additional labelling was found in pre-SMA, CMA-r and ventral Pfc (Fig. 6). Note that labelling was observed in dorsal premotor areas (Fig. 6, section 22), but this projection was not confirmed in the other cases with similar injections. Finally, there was no labelling in PMv-c, i.e. behind the genu.
Injections in PMv-c
Five injections were made in PMv-c (Table I). These injections gave rise to strong labelling in the contralateral frontal areas, with the core of labelling in PMv-c and PMv-r in all cases. Figures 5 and 6 illustrate two representative examples (green dots). As in the cases with injections in PMv-r, callosal labelling following injections in PMv-c is located mainly lateral to the genu of the arcuate sulcus and in mesial cortex. The most extensive labelling was found in the ventral premotor region (including both PMv-r and PMv-c), where it spanned the cortex caudal and anterior to the level of the genu of the arcuate sulcus (sections 18–38 in Fig. 5 and 6). Weak labelling was found consistently in pre-SMA, CMA-r and M1, and in some cases in PMd-r, SMA-proper, CMA-v and CMA-d and PMd-c (see Fig. 8).
Comparison between PMd and PMv
The present study allowed a direct comparison between the callosal connections of the four premotor areas investigated. Comparison can be made directly on coronal sections in figures 5 and 6 (two monkeys with large injections of 4 tracers each) and on a 2D map of the cortex in figure 7, in monkey Mk6 where we made small injections of 3 tracers (see also Fig. 8).
It appears that, at a gross level, callosal projections to dorsal and ventral premotor sectors are organized along both the rostro-caudal and the medio-lateral axes (Fig. 5 and 6). Along the rostro-caudal axis, injections in rostral sectors (PMd-r and PMv-r) tend to yield stronger labelling in rostral frontal areas of the opposite hemisphere, i.e. anterior to the level of the genu of the arcuate sulcus (see for example Fig. 6; red and grey dots). Similarly, large injections in the caudal sectors (PMd-c and PMv-c) resulted in strong callosal labelling in caudal frontal areas (blue and green dots), with however, important labelling in rostral regions overlapping with the projections to rostral sectors. This might be due to the large size of the injection sites, as small injections into PMd-c and PMv-c (Fig. 7) led to less overlap. A similar pattern of labelling is also observed following injections into PMv-c (green dots in Fig. 5 and 6). Figure 8A shows the percentage of cells in different areas, organized a rostral and a caudal group.
Superimposed to this trend, the organisation of callosal projections along the medio-lateral axis is even more striking. Figures 5 and 6 show that callosal inputs to PMd arise almost exclusively from the dorso-medial regions (red and blue dots), whereas those to PMv originate predominantly from lateral regions (green and grey dots), with small zones of overlap in pre-SMA, the cingulate motor areas, the border between PMd-c and PMv-c, and the most medial part of PMd-r. Figure 7 illustrates more clearly the topography of callosal labelling after three small injections in monkey Mk6.
A quantitative analysis was conducted on data from 11 injections in 3 monkeys (Mks 1–3) following the procedure described in the Methods section. This procedure provided a numerical estimate of the contribution of each area to the overall callosal afferent connectivity of PMd-r, PMd-c, PMv-r and PMv-c. The results of this analysis are represented graphically for each monkey in Figure 8A (see also [1 ]).
Figure 8A shows the variability across the animals with large injections of tracers in the four premotor sectors (Mks 1–3). The variability appeared most prominently for the percentage of the homotopic callosal projections, which ranged from 29 to 40% for PMd-r (in 2 monkeys), from 43 to 81% for PMv-r, from 16 to 23% for PMd-c and from 14 to 19% for PMv-c. For the two latter divisions, the variability was larger for the percentage of the inputs coming from the heterotopic contralateral PMv-r (Fig. 8A). Despite this variability, it appears that PMd-r and PMv-r receive most of their callosal inputs from rostral frontal areas, especially for PMd-r; Inputs from caudal regions are weak or absent. By contrast, PMd-c and, to a lesser extent, PMv-c receive inputs from both rostral and caudal areas.
Figure 8B compares the results of the present study with those previously reported for pre-SMA and SMA-proper , using the same analysis of percent contribution of contralateral frontal areas. It appears that the variability observed here is in the same order of magnitude as that observed among 4 monkeys with injections in pre-SMA and SMA-proper.