- Email: [email protected]
Spatial distribution of cingulate cells projecting to the primary, supplementary, and pre-supplementary motor areas: a retrograde multiple labeling study in the macaque monkey
Neuroscience Research 39 (2001) 39 – 49 www.elsevier.com/locate/neures
Spatial distribution of cingulate cells projecting to the primary, supplementary, and pre-supplementary motor areas: a retrograde multiple labeling study in the macaque monkey Yan Wang a,b, Keisetsu Shima a, Hiromasa Sawamura a, Jun Tanji a,b,* a
Department of Physiology, Tohoku Uni6ersity School of Medicine, Seiryo-cho, Aobaku, Sendai 980, Japan b Core Research for E6olutional Science and Technology Program, Kawaguchi 332 -0012, Japan Received 2 August 2000; accepted 11 September 2000
Abstract We examined the location and spatial distribution of cingulate cortical cells projecting to the forelimb areas of the primary motor cortex (MI), supplementary motor area (SMA), and pre-supplementary motor area (pre-SMA) using a multiple retrograde labeling technique in the monkeys (Macaca fuscata). The forelimb areas of the MI, SMA and pre-SMA were physiologically identified, based on the findings of intracortical microstimulation (ICMS) and single cell recording. Three different tracers, diamidino yellow (DY), fast blue (FB), and wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), were injected into each of the three motor areas in the same monkey. Retrogradely labeled cells in the cingulate cortex were plotted with an automated plotting system. Cells projecting to the forelimb area of the MI were distributed in the two separate regions situated rostrocaudally in the dorsal and ventral banks of the cingulate sulcus, namely the rostral cingulate motor area (CMAr) and caudal cingulate motor area (CMAc). These two regions corresponded to the forelimb areas identified by the ICMS in the same animal. The distribution of projection cells to the SMA overlapped extensively with that of projection cells to the MI. Although the MI received relatively sparse inputs from the CMAr than from the CMAc, the SMA received inputs from the CMAr and its adjacent areas as much as from the CMAc. The projection cells to the pre-SMA were distributed in the anterior portion of the cingulate cortex, including the anterior part of the CMAr and in a small part of the cingulate gyrus. These findings indicate that the MI and SMA share a considerable common information from the cingulate cortex, including the CMAr and CMAc, whereas the pre-SMA receives a different set of information from the anterior part of the cingulate cortex. © 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Cingulate motor cortex; Primary motor cortex; Supplementary motor area; Pre-supplementary motor area; Primate; Retrograde labeling
1. Introduction It has been established that two separate motor representation areas exist in the cingulate cortex, termed the rostral and caudal cingulate motor areas (CMAr and CMAc, Dum and Strick, 1991; Luppino et al., 1991; Shima et al., 1991). These two areas, known to have somatotopic organization (Dum and Strick, * Corresponding author. Tel.: +81-22-7178071; fax: + 81-227178077. E-mail address: [email protected] (J. Tanji).
1991; Luppino et al., 1991; He et al., 1995) and to project to the primary motor cortex (MI, Muakkassa and Strick, 1979), constitute a limited portion within the cingulate cortex (Picard and Strick, 1996). It was also reported that both the supplementary motor area (SMA) and the pre-supplementary motor area (preSMA) receive inputs from the cingulate cortex (Morecraft and Van Hoesen, 1992; Luppino et al., 1993). These studies led to the next questions of how extensively the projections into the three areas (MI, SMA, and pre-SMA) overlap, and how much of the cingulate projections to the SMA and pre-SMA arise from within
0168-0102/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 0 0 ) 0 0 1 9 8 - X
Y. Wang et al. / Neuroscience Research 39 (2001) 39–49
40
or outside of the CMAr and CMAc, defined as in previous reports. The present study was designed to answer the questions with a technique of multiple labeling of projection cells in monkeys whose motor areas are physiologically identified (Shindo et al., 1995). The results will show territories of cingulate projection zones to the three motor areas, which suggest different sets of information provided from separate portions of the cingulate cortex. A preliminary account of this study has appeared elsewhere (Wang et al., 1999).
2. Materials and methods Four Japanese monkeys (Macaca fuscata) weighing 5 – 7.5 kg were used in this study. The animals were cared for in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals, and Guidelines for Institutional Animal Care and Use published by our institute. After they were fully accustomed to sitting quietly in a primate chair with their limbs restrained during electrophysiolgical examination, they were anesthetized for surgery with ketamine hydrochloride (5 mg/kg, i.m.) and sodium pentobarbital (30 mg/kg, i.m.). Ketamine was added as needed. Under aseptic conditions, the skull was removed adequately to expose the MI, SMA, and preSMA. The dura mater was left intact. Acrylic recording chambers and head fixation bolts were implanted on the skull. After the surgery, monkeys were given antibiotics and returned to their cages. After a 1-week recovery period, electrophysiological examinations began. For intracortical microstimulation (ICMS) and for recording extracellular unit activity, we used glass-insulated Elgiloy microelectrodes with measured impedance of 0.8–1.0 MV (at 300 Hz). For stimulation, trains of 12 cathodal 200 ms pulses were applied at 333 Hz
through a constant current stimulator. Current intensities routinely ranged from 3 to 40 mA. However, 22 or 42 pulses and currents of up to 50 mA were also used for the pre-SMA and cingulate cortex, and for some parts of the SMA. Physiological examinations were performed in fully awake monkeys. ICMS was begun when the microelectrode reached a depth of 1.0 mm from the pial surface and, thereafter, was performed at every 500 mm of electrode advancement. In each area, we mapped the topographical organization based on ICMS effects, and on cell receptive fields of somatosensory responses. After identifying the forelimb areas of the SMA, preSMA, MI and cingulate cortex, electrolytic microlesions (anodal currents of 350 mC) were placed to use as reference points for later reconstruction of electrode tracks. Microlesions were made at the deepest points of microelectrode entry. Cortical depths at which ICMS effects were determined were calculated on the basis of depth reading by the stepping microdrive. Three different tracers were used: 5% diamidino yellow (DY, Sigma; dissolved in saline), 5% fast blue (FB, Sigma; dissolved in saline), and 5% wheat germ agglutinin conjugated to horseradish peroxidase (WGAHRP, Toyobo; dissolved in 0.5 M NaCl with 0.1 M mannose). On the basis of the map constructed by the electrophysiological studies, we selected the sites for tracer injections in the SMA, pre-SMA, and MI. The choice of tracers at each injection site and the amount injected in each animal are listed in Table 1. Tracers were pressure-injected at one or two depths with a 1.0-ml Hamilton syringe attached to the same manipulator as used for microelectrode insertion. The amount per injection was 0.3–0.4 ml for DY and FB, and 0.1–0.15 ml for WGA-HRP. The survival period was 3 weeks for fluorescent dyes (DY and FB) and 3 days for WGA-HRP. The survival period after each injection
Table 1 Injection site and tracers injecteda Monkey No.
Injection sites
Tracer
Number of injection sites
Total amount (ml)
1
MI SMA Pre-SMA
WGA-HRP DY FB
3 1 1
0.75 0.80 0.80
2
MI SMA Pre-SMA
WGA-HRP FB DY
3 1 1
0.75 0.80 0.80
3
MI Pre-SMA
WGA-HRP DY
3 1
1.20 0.80
4
MI SMA Pre-SMA
WGA-HRP DY FB
4 1 1
1.05 0.80 0.80
a Injection sites are abbreviated as MI, the forelimb area of the primary motor cortex; SMA, the forelimb area of the supplementary motor area; pre-SMA, the forelimb area of the presupplementary motor area.
Y. Wang et al. / Neuroscience Research 39 (2001) 39–49
41
Fig. 1. Photomicrographs showing injection sites (A, B and C) and labeled cells in the cingulate motor area (D and E). (A) diamidino yellow (DY) injection into the supplementary motor area (SMA). (B) Fast blue (FB) injection into the pre-supplementary motor area (pre-SMA). (C) Wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) injection into the primary motor cortex (MI). (D) FB- and DY-labeled cells under epifluorescent illumination. FB-labeled cells appear as blue spots. Arrows indicate DY-labeled cells. The nuclear localization of DY and the cytosolic localization of FB can be seen. (E) WGA-HRP-labeled cells under the mid-condition of brightfield and epifluorescent illumination in the same section. The dashed lines in D and E illustrate the borders of adjoining cortical layers (III, V, and VI). Calibration bars in A – C are 1 mm.
42
Y. Wang et al. / Neuroscience Research 39 (2001) 39–49
was found adequate for detecting corticocortical projection cells in M. fuscata. Fig. 1A – C shows photomicrographs of injection sites of fluorescent dyes and WGA-HRP. Under deep anesthesia with ketamine hydrochloride (5 mg/kg, i.m.) and sodium pentobarbital (50 mg/kg, i.m.), each monkey was perfused transcardially with 0.1 M phosphate buffer (pH 7.4) containing heparin. Fixative solutions were then perfused in the following order: 3% paraformaldehyde in 0.1 M phosphate buffer, 3% paraformaldehyde in 0.1 M phosphate buffer with 10% glycerin, 0.1 M phosphate buffer with 10% glycerin and 0.1 M phosphate buffer with 20% glycerin. The brains were removed, blocked, and for a few days before sectioning they were stored at 4°C in a solution containing 20% glycerin and 2% dimethyl sulfoxide in 0.1 M phosphate buffer (pH 7.4). Serial sections were made at 50 mm in the frontal plane on a freezing microtome. Each section was mounted on a gelatin-coated glass slide. Every alternate section was left unstained for examination with a fluorescence microscope, and was later Nissl-stained with cresyl violet for cytoarchitectonic examination. The remaining half of the sections was processed for HRP staining with tetramethylbenzidine. As shown in Fig. 1D and E, cells labeled with fluorescent dyes (DY and FB) can be clearly seen even in HRP-processed sections (Shindo et al., 1995). It was possible to examine cells labeled with WGA-HRP and with DY (or FB) in the single sections under both brightfield and epifluorescent illumination with 360-nm ultraviolet excitation. Thus, plotting was made in HRPprocessed sections using a computerized digital plotting system (MD2 Microscope Digitizer, Minnesota Digitizer, Minnesota Datametrics) to measure their locations in each section. In every fourth HRP-processed section, all labeled cells were plotted to examine the spatial distribution of projection cells in the cingulate cortex. WGA-HRP injection sites were considered to include the densely stained regions adjacent to the needle track and the surrounding halo. DY and FB injection sites were considered to include the first and second zones of Keizer et al. (1983). Each injection site was reconstructed from serial coronal sections with systematic measurements of its extent and spatial relation to the surrounding structures. To measure the density of projection cells, we performed a quantitative analysis of labeled cells. In each coronal section, the gray matter of the cortex in the mesial hemisphere above the corpus callosum was divided into unitary compartments (laminae of 500-mm width), perpendicular to the cortical surface. The number of labeled cells with each tracer was counted in each 500-mm lamina, using the computerized counting system.
3. Results
3.1. Physiological identification of injection sites The forelimb representation area of the SMA was identified by a combination of the following criteria: (1) ICMS evoked forelimb movements with thresholds of less than 30 mA; (2) cell activity related to the animal’s voluntary forelimb movements; (3) somatosensory responses evoked by touching the forelimb or manipulating forelimb joints; and (4) absence of visually evoked responses (Tanji and Kurata, 1979; Macpherson et al., 1982; Mitz and Wise, 1987; Tanji, 1994). A representative example of topographical mapping in the forelimb area of the SMA is shown in Fig. 2. Rostral to the forelimb area, the face was represented. Further rostrally, a separate forelimb area was found. The rostral area corresponded to the pre-SMA according to the following criteria: (1) ICMS-evoked complex forelimb
Fig. 2. Schematic diagram showing results of electrophysiological mapping and injection sites of tracers in the SMA and pre-SMA of monkey c 1. (Top) Top view of the left hemisphere showing the location of cortical injection sites (*). (Middle) Each label indicates a penetration site where movements were elicited by the intracortical microstimulation (ICMS) in the following body parts: Fa, face; El, elbow; Wr, wrist; D, digits; T, trunk; H, hindlimb. (Bottom) Based on histological examinations, representative electrode tracks at penetrations in and around injection sites are reconstructed (El1-El2, D3-El4), along with the directions indicated with interrupted lines. Evoked movements by the ICMS are shown at about 500-mm depths of the electrode tracks. DY and FB injections were made into the forelimb area of the SMA and pre-SMA, respectively. The spread of tracers is indicated by dark and light areas corresponding to the first and second zones of Keizer et al. (1983). Crosses denote injection points. CS, central sulcus; ARC, arcuate sulcus; PS, principle sulcus. Note that each tracer was confined within the forelimb representation area. A thin arrow indicates the caudal end of arcuate sulcus.
Y. Wang et al. / Neuroscience Research 39 (2001) 39–49
movements with higher threshold of 30 – 50 mA; (2) cell activity related to the animal’s voluntary forelimb movements; (3) little or no somatosensory responses to natural stimuli; and (4) presence of visual responses to flash of lights or moving objects (Luppino et al., 1991; Matsuzaka et al., 1992). FB or DY was injected into the central focus of the forelimb area of the SMA or pre-SMA at depths of 1.0 – 4.0 mm from the pial surface. The spread of injections was no larger than 2 mm in diameter, and was confined to the forelimb representation within the gray matter of the SMA and preSMA. The forelimb areas of the MI were identified by the ICMS and somatosensory responses (Sessle and Wiesendanger, 1982; Sato and Tanji, 1989; Huntley and Jones, 1991). We surveyed extensively the entire MI, both the rostral bank of the central sulcus and the surface of the precentral gyrus. A representative example of topographical mapping in the MI observed in monkey c1 is shown in Fig. 3. Multiple injections were made into the forelimb area of the MI. Injections were placed at depths of 1 – 1.5 and 2 – 2.5 mm from the pial surface in the rostral bank of the central sulcus, and at depths of 1–1.5 mm from the pial surface in the cranial part of the precentral gyrus. The spread of
Fig. 3. Schematic diagram showing results of electrophysiological mapping and injection sites of a tracer in the MI of monkey c1. (Top-right) top view of the left hemisphere showing the location of cortical injection sites (asterisks). (Bottom) precentral sites of microelectrode entry. Each label indicates a penetration site where movements were elicited from more than a half of the ICMS delivery in the following body parts: Fa, face; Ne, neck; D, digits; Wr, wrist; El, elbow; Sh, shoulder. Based on histological examinations, representative electrode tracks at penetrations around injection sites are reconstructed (El1-Wr2, El3-Wr4, D5-D6), along with the directions indicated with interrupted lines and drawn. Evoked movements by the ICMS are shown at about 500-mm depth of the electrode tracks. Three WGA-HRP injections were made into the forelimb area of the MI. The spread of tracers is indicated by shaded areas with dark and light areas. Crosses denote injection points. CS, central sulcus; ARC, arcuate sulcus; PS, principle sulcus.
43
injections was within 2.5 mm in diameter, and confined to each representation within the gray matter of the MI. Later histological analysis showed that injections were localized within cytoarchitectonic area 4 of Brodmann characterized by the presence of giant pyramidal cells in layer V. Frontal reconstructions through the three injection sites are illustrated in Fig. 3.
3.2. Distribution of retrogradely labeled cingulate cells Three different tracers were injected into the forelimb area of the MI, SMA and pre-SMA of three monkeys and two different tracers were injected into the forelimb area of the MI and pre-SMA in one monkey (Table 1). Among them, one monkey was used for the ICMS mapping of the cingulate motor areas as well as the SMA and pre-SMA. Fig. 4 shows the distribution of labeled cells in an unfolded map of the medial surface of the hemisphere, and the topographical motor map obtained by the ICMS (monkey c4). In this monkey, WGA-HRP was injected into the MI, and DY and FB were injected into the SMA and pre-SMA, respectively (Table 1). As shown in Fig. 4, two clusters of WGA-HRP labeled cells were observed in the cingulate cortex rostrocaudally in the banks of the cingulate sulcus. The large caudal cluster composed of densely packed labeled cells was located at the dorsal and ventral banks of the cingulate sulcus in a region termed areas 6c and 23c by Dum and Strick (1991), LC by Von Bonin and Bailey (1947), and area 23c by Vogt et al. (1987). The rostral cluster of labeled cells was located at the rostral portion of cingulate cortex corresponding to FDL by Von Bonin and Bailey (1947), and area 24c by Vogt et al. (1987). This cluster was found in the ventral and dorsal banks of the cingulate sulcus at the level of the pre-SMA and SMA. In this report, we called the rostral and caudal foci of MI-projection cells as the CMAr and CMAc. A cluster of HRP-labeled cells was also found in the SMA, but not in the pre-SMA. In the monkey c 4, DY-labeled cells projecting to the SMA were widely distributed over the medial wall of the hemisphere, with several clusters. Some clusters of DY-labeled cells overlapped with the CMAr (Fig. 5C) and CMAc (Fig. 5D). The overlap was particularly extensive in the CMAc. In contrast, FB-labeled cells projecting to the pre-SMA were distributed in the rostral part of the cingulate cortex and in the superior frontal gyrus rostral to the injection site. Although FB-labeled cells showed partial overlap with the rostral part of the CMAr, few FB-labeled cells were observed in and around the CMAc. Fig. 4C shows the topographic motor map obtained by the ICMS in the same monkey. Generally, the areas where forelimb movements were evoked corresponded to regions in which cells project to the forelimb areas of
44
Y. Wang et al. / Neuroscience Research 39 (2001) 39–49
Fig. 4. Plots of three different retrogradely labeled cells on the medial wall of hemisphere in monkey c4 (B) and the topographical motor map in the same region by the ICMS (C). (A) Top view of the left hemisphere showing the location of cortical injection sites (asterisks). CS, central sulcus; ARC, arcuate sulcus; PS, principle sulcus; Cing. S, cingulate sulcus; CC, corpus callosum. The letters a, b, c, and d in the schematic section drawing inset at top correspond to a, b, c, and d in B and C. Arrows in B and C indicate the caudal end of arcuate sulcus. In B, each dot represents two to five labeled cells.
Y. Wang et al. / Neuroscience Research 39 (2001) 39–49
45
Fig. 5. Distribution of retrogradely labeled cells in monkey c1. Each dot represents two to five labeled cells. (Top) top view of the left hemisphere showing the location of cortical injection sites (asterisks). The approximate positions of the coronal sections are specified by lines labeled with A to D. (Middle and Bottom) Coronal sections are arranged from A to D. Green, FB-labeled cells; blue, DY-labeled cells; red, WGA-HRP-labeled cells.
the MI, namely CMAr and CMAc. The hindlimb movements were elicited in the areas located caudal to the CMAc and in a region between the CMAr and CMAc. Fig. 5 shows the distribution of labeled cells in coronal sections obtained at four rostro-caudal levels (monkey c1). WGA-HRP-labeled and DY-labeled cells were overlapping extensively in the banks of the cingulate sulcus, both in the CMAr (Fig. 5C) and CMAc (Fig. 5D). However, in overlapping regions, the HRP- and DY-labeled cells were interdigitated, rather
than intermixed. The number of double-labeled cells was less than 2% of labeled cells in the CMAr and CMAc. As shown in Fig. 5A and B, FB-labeled cells were concentrated around the cingulate sulcus of the anterior cingulate cortex.
3.3. Quantitati6e analysis of distribution of projection cells To determine more precisely the spatial distribution and density of projection cells to the MI, SMA, and
46
Y. Wang et al. / Neuroscience Research 39 (2001) 39–49
pre-SMA, quantitative analysis was made on the basis of data obtained in three monkeys. As mentioned in Section 2, the cingulate cortex in each section was divided into unitary compartments of 500-mm laminae perpendicular to the cortical surface. In each lamina, the numbers of labeled cells with the three tracers were counted and plotted on the unfolded medial surface map of the hemisphere. The density map obtained from the data in monkey c 1 is shown in Fig. 6. Two clusters of labeled cells projecting to the forelimb area of the MI were observed rostocaudally along the cingulate sulcus, as in Fig. 4B. The areal size of the CMAc was larger than that of the CMAr. The density of WGA-HRP labeled cells in the CMAc (8.89: mean number of labeled cells/lamina) was
significantly higher (P B 0.001, t-test) than that in the CMAc (4.74/lamina). Labeled cells projecting to the forelimb area of the SMA were widely distributed in the cingulate cortex. However, high-density regions were found in the CMAr and CMAc and their adjacent regions. The mean density of labeled cells projecting to the SMA was greater (PB 0.001, t-test) in the CMAr (17.61/lamina) than in the CMAc (11.07/lamina). Projection cells to the preSMA were mainly located in the rostral part of the cingulate cortex and in a small part of the cingulate gyrus. These regions showed partial overlap with the region projecting to the SMA and ventral part of the CMAr, but showed little overlap with the CMAc.
Fig. 6. Quantified plots of three different retrogradely labeled cells on the medial wall of hemisphere in monkey c1. (Top) top view of the left hemisphere showing the location of cortical injection sites (*). In this figure and in Fig. 6, red, blue and green bars indicate projection cells to the MI, SMA and pre-SMA, respectively. Sizes of bars indicate the density of labeled cells in each bin (see Section 2).
Y. Wang et al. / Neuroscience Research 39 (2001) 39–49
47
4. Discussion
Fig. 7. Frequencies of occurrences of unitary compartments (laminae) within the CMAr (left) and CMAc (right), possessing labeled cells projecting to the SMA and/or pre-SMA, in addition to cells projecting to the MI. The data obtained from three monkeys (c 1, 2 and 4) were averaged and expressed as percentages.
The quantitative analysis was performed on the data obtained from three monkeys, giving rise to basically the same results as in monkey c1. To summarize the degree of overlap of projections to the three areas, we first analyzed how many of the unitary compartments (individual laminae) in the CMAr and CMAc contained labeled cells projecting to the SMA and/or preSMA, in addition to projection cells to the MI. For this purpose, we calculated the average number of compartments including labeled cells in various combinations, from the data for the three monkeys. Fig. 7 shows the frequency of occurrences of the compartments, expressed as percentages. In both the CMAr and CMAc, a majority of the compartments (57 and 71%, respectively) projects to the SMA, as well as to the MI. In the CMAr, 19% of the compartments included cells projecting to all three areas. In contrast, in the CMAc, only a small minority of compartments possessed cells projecting to the pre-SMA. Subsequently, we examined the degree of overlap within the areas projecting to the SMA and to the pre-SMA, as shown in Fig. 8. Thirty four percent of the pre-SMA-projecting compartments included SMA-projecting cells, whereas 16% of the SMA-projecting compartments included pre-SMA-projecting cells. The compartments with projection cells to the pre-SMA and MI were small in number, compared with compartments with shared projections to the SMA and MI. The great majority of pre-SMA-projecting compartments (85%) did not include MI-projection cells.
In this study we investigated the spatial distribution of cingulate cells projecting to the forelimb areas in the MI, SMA, and pre-SMA, with a technique of injecting three different tracers into physiologically identified motor areas. We found that both the CMAr and CMAc, defined as cingulate motor areas projecting to the MI, have extensive projections to the SMA. In contrast, only a small fraction of cingulate motor areas contained cells projecting to the pre-SMA. Especially, the projection from the CMAc to the pre-SMA was meager. The cingulate areas projecting to the pre-SMA and SMA exhibited overlap in 34% of pre-SMA-projecting territory and in 16% of SMA-projecting territory. On the other hand, about a half of the SMA-projecting area, and also a half of the pre-SMA projecting area included no cells projecting to other motor areas. These findings indicate that the MI and SMA receive common sources of information from the cingulate motor areas, whereas the cingulate inputs for the MI and pre-SMA are largely separate. Furthermore, the input sources for the pre-SMA and SMA arise primarily from the rostral and caudal parts of the cingulate motor areas, with a partial overlap. It seems important, however, to acknowledge limitations in interpreting our present data, although the forelimb representation areas were physiologically identified and tracer injections were made in a portion of each area. We have to be cautious about possible underestimates of the number and distribution of projection cells. It should also be mentioned that the tracer injections into hindlimb or facial representation areas are likely to reveal different sets of projection cells (Morecraft and Van Hoesen, 1993; Morecraft et al., 1996; Tokuno et al., 1997).
Fig. 8. Frequencies of occurrences of unitary compartments projecting to the pre-SMA (left) and SMA (right), possessing a variety of combinations of cells projecting to other three motor areas.
48
Y. Wang et al. / Neuroscience Research 39 (2001) 39–49
The present study confirmed previous reports (Luppino et al., 1993; Morecraft and Van Hoesen, 1993; He et al., 1995;) in that the density of projection cells to the MI is larger in the CMAc than in the CMAr. We further found in this study that the CMAr and its adjacent regions densely project to the SMA. Further rostrally, the banks of the cingulate sulcus project to the pre-SMA without much projection to other motor areas. These findings suggest that the rostral part of the cingulate cortex is heterogeneous with respect to efferent projections, while the caudal part appears more homogeneous with prominent outputs to the MI and SMA. A previous study from our laboratory (Shima et al., 1991) found that the cell activity in the caudal cingulate motor area showed resemblance to that in the MI, primarily in relation to execution of movements. On the other hand, cells in the rostral cingulate motor area exhibited relations to motor preparation (Shima et al., 1991) or to reward-based motor selection (Shima and Tanji, 1998). Very recently, anterior cingulate cells were found to be active preferentially in relation to routine or non-routine sequential motor behavior (Procyk et al., 2000). These single-cell recording studies imply the role of rostral cingulate cortex in cognitive aspects of motor behavior. This view finds support from a large number of brain imaging studies in human subjects (Carter et al., 1998; Petit et al., 1998; Kiehl et al., 2000; for review, see Picard and Strick, 1996). Considering the diversity of outputs from the rostral cingulate cortex as found in this study, it is necessary in future studies to determine whether various aspects of cell activity are specialized in certain areas with specific output projections.
Acknowledgements We thank M. Kurama and Y. Takahashi for technical assistance. The work was supported by grants from the Ministry of Education, Science, and Culture of Japan (08 279 101 and 09 308 032), and by Japan Science and Technology Corporation.
References Carter, C.S., Braver, T.S., Barch, D.M., Botvinick, M.M., Noll, D., Cohen, J.D., 1998. Anterior cingulate cortex, error detection, and the online monitoring of performance. Science 280, 747–749. Dum, R.P., Strick, P.L., 1991. The origin of corticospinal projections from the premotor areas in the frontal lobe. J. Neurosci. 11, 667 – 689. He, S.Q., Dum, R.P., Strick, P.L., 1995. Topographic organization of corticospinal projections from the frontal lobe: motor areas on the medial surface of the hemisphere. J. Neurosci. 15, 3284 – 3306.
Huntley, G.W., Jones, E.G., 1991. Relationship of intrinsic connections to forelimb movement representations in monkey motor cortex: a correlative anatomical and physiological study. J. Neurophysiol. 66, 390 – 413. Keizer, K., Kuypers, H.G.J.M., Huisman, A.M., Dann, O., 1983. Diamidino yellow dihydrochloride (DY:2HCl): a fluorescent retrograde neuronal tracer which migrates only very slowly out of the cell and can be used in combination with TB and FB in double labeling experiments. Exp. Brain Res. 51, 179 – 191. Kiehl, K.A., Liddle, P.F., Hopfinger, J.B., 2000. Error processing and the rostral anterior cingulate: an event-related fMRI study. Psychophysiology 37, 216 – 223. Luppino, G., Matelli, M., Camarda, R.M., Gallese, V., Rizzolatti, G., 1991. Multiple representations of body movements in mesial area 6 and the adjacent cingulate cortex: an intracortical microstimulation study in the macaque monkey. J. Comp. Neurol. 311, 463 – 482. Luppino, G., Matelli, M., Camarda, R., Rizzolatti, G., 1993. Corticocortical connections of area F3 (SMA-proper) and area F6 (pre-SMA) in the macaque monkey. J. Comp. Neurol. 338, 114– 140. Macpherson, J.M., Marangoz, C., Miles, T.S., Wiesendanger, M., 1982. Microstimulation of the supplementary motor area (SMA) in the awake monkey. Exp. Brain Res. 45, 410 – 416. Matsuzaka, Y., Aizawa, H., Tanji, J., 1992. A motor area rostral to the supplementary motor area (presupplementary motor area) in the monkey: neuronal activity during a learned motor task. J. Neurophysiol. 68, 653 – 662. Mitz, A.R., Wise, S.P., 1987. The somatotopic organization of the supplementary motor area: intracortical microstimulation mapping. J. Neurosci. 7, 1010 – 1021. Morecraft, R.J., Van Hoesen, G.W., 1992. Cingulate input to the primary and supplementary motor cortices in the rhesus monkey: evidence for somatotopy in areas 24c and 23c. J. Comp. Neurol. 322, 471 – 489. Morecraft, R.J., Van Hoesen, G.W., 1993. Frontal granular cortex input to the cingulate (M3), supplementary (M2) and primary (MI) motor cortices in the rhesus monkey. J. Comp. Neurol. 337, 669 – 689. Morecraft, R.J., Schroeder, C.M., Keifer, J., 1996. Organization of face representation in the cingulate cortex of the rhesus monkey. Neuroreport 7, 1343 – 1348. Muakkassa, K.F., Strick, P.L., 1979. Frontal lobe inputs to primate motor cortex: evidence for four somatotopically organized ’premotor’ areas. Brain Res. 177, 176 – 182. Petit, L., Courtney, S.M., Ungerleider, L.G., Haxby, J.V., 1998. Sustained activity in the medial wall during working memory delays. J. Neurosci. 18, 9429 – 9437. Picard, N., Strick, P.L., 1996. Motor areas of the medial wall: a review of their location and functional activation. Cereb. Cortex 6, 342 – 353. Procyk, E., Tanaka, Y.L., Joseph, J.P., 2000. Anterior cingulate activity during routine and non-routine sequential behaviors in macaques. Nat. Neurosci. 3, 502 – 508. Sato, K.C., Tanji, J., 1989. Digit-muscle responses evoked from multiple intracortical foci in monkey precentral motor cortex. J. Neurophysiol. 62, 959 – 970. Sessle, B.J., Wiesendanger, M., 1982. Structural and functional definition of the motor cortex in the monkey (Macaca fascicularis). J. Physiol. (London) 323, 245 – 265. Shima, K., Tanji, J., 1998. Role for cingulate motor area cells in voluntary movement selection based on reward. Science 282, 1335 – 1338. Shima, K., Aya, K., Mushiake, H., Inase, M., Aizawa, H., Tanji, J., 1991. Two movement-related foci in the primate cingulate cortex observed in signal-triggered and self-paced forelimb movements. J. Neurophysiol. 65, 188 – 202.
Y. Wang et al. / Neuroscience Research 39 (2001) 39–49 Shindo, K., Shima, K., Tanji, J., 1995. Spatial distribution of thalamic projections to the supplementary motor area and the primary motor cortex: a retrograde multiple labeling study in the macaque monkey. J. Comp. Neurol. 357, 98–116. Tanji, J., Kurata, K., 1979. Neuronal activity in the cortical supplementary motor area related with distal and proximal forelimb movements. Neurosci. Lett. 12, 201–206. Tanji, J., 1994. The supplementary motor area in the cerebral cortex. Neurosci. Res. 19, 251–268. Tokuno, H., Takada, M., Nambu, A., Inase, M., 1997. Reevaluation of ipsilateral corticocortical inputs to the orofacial region of the primary motor cortex in the macaque monkey. J. Comp. Neurol.
.
49
389, 34 – 48. Vogt, B.A., Pandya, D.N., Rosene, D.L., 1987. Cingulate cortex of the rhesus monkey: I. Cytoarchitecture and thalamic afferents. J. Comp. Neurol. 262, 256 – 270. Von Bonin, G., Bailey, P., 1947. The neocortex of Macaca mulatta. In: Monographs in Medical Sciences, vol. 5. University of Illinois Press, Urbana, IL, p. 136. Wang, Y., Shima, K., Sawamura, H., Tanji, J., 1999. A multiple labeling study of projection neurons in the cingulate cortex to the primary motor cortex, the supplementary motor area, and the pre-supplementary motor area. Neurosci. Res. 23, S199.
Spatial distribution of cingulate cells projecting to the primary, supplementary, and pre-supplementary motor areas: a retrograde multiple labeling study in the macaque monkey Yan Wang a,b, Keisetsu Shima a, Hiromasa Sawamura a, Jun Tanji a,b,* a
Department of Physiology, Tohoku Uni6ersity School of Medicine, Seiryo-cho, Aobaku, Sendai 980, Japan b Core Research for E6olutional Science and Technology Program, Kawaguchi 332 -0012, Japan Received 2 August 2000; accepted 11 September 2000
Abstract We examined the location and spatial distribution of cingulate cortical cells projecting to the forelimb areas of the primary motor cortex (MI), supplementary motor area (SMA), and pre-supplementary motor area (pre-SMA) using a multiple retrograde labeling technique in the monkeys (Macaca fuscata). The forelimb areas of the MI, SMA and pre-SMA were physiologically identified, based on the findings of intracortical microstimulation (ICMS) and single cell recording. Three different tracers, diamidino yellow (DY), fast blue (FB), and wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), were injected into each of the three motor areas in the same monkey. Retrogradely labeled cells in the cingulate cortex were plotted with an automated plotting system. Cells projecting to the forelimb area of the MI were distributed in the two separate regions situated rostrocaudally in the dorsal and ventral banks of the cingulate sulcus, namely the rostral cingulate motor area (CMAr) and caudal cingulate motor area (CMAc). These two regions corresponded to the forelimb areas identified by the ICMS in the same animal. The distribution of projection cells to the SMA overlapped extensively with that of projection cells to the MI. Although the MI received relatively sparse inputs from the CMAr than from the CMAc, the SMA received inputs from the CMAr and its adjacent areas as much as from the CMAc. The projection cells to the pre-SMA were distributed in the anterior portion of the cingulate cortex, including the anterior part of the CMAr and in a small part of the cingulate gyrus. These findings indicate that the MI and SMA share a considerable common information from the cingulate cortex, including the CMAr and CMAc, whereas the pre-SMA receives a different set of information from the anterior part of the cingulate cortex. © 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Cingulate motor cortex; Primary motor cortex; Supplementary motor area; Pre-supplementary motor area; Primate; Retrograde labeling
1. Introduction It has been established that two separate motor representation areas exist in the cingulate cortex, termed the rostral and caudal cingulate motor areas (CMAr and CMAc, Dum and Strick, 1991; Luppino et al., 1991; Shima et al., 1991). These two areas, known to have somatotopic organization (Dum and Strick, * Corresponding author. Tel.: +81-22-7178071; fax: + 81-227178077. E-mail address: [email protected] (J. Tanji).
1991; Luppino et al., 1991; He et al., 1995) and to project to the primary motor cortex (MI, Muakkassa and Strick, 1979), constitute a limited portion within the cingulate cortex (Picard and Strick, 1996). It was also reported that both the supplementary motor area (SMA) and the pre-supplementary motor area (preSMA) receive inputs from the cingulate cortex (Morecraft and Van Hoesen, 1992; Luppino et al., 1993). These studies led to the next questions of how extensively the projections into the three areas (MI, SMA, and pre-SMA) overlap, and how much of the cingulate projections to the SMA and pre-SMA arise from within
0168-0102/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 0 0 ) 0 0 1 9 8 - X
Y. Wang et al. / Neuroscience Research 39 (2001) 39–49
40
or outside of the CMAr and CMAc, defined as in previous reports. The present study was designed to answer the questions with a technique of multiple labeling of projection cells in monkeys whose motor areas are physiologically identified (Shindo et al., 1995). The results will show territories of cingulate projection zones to the three motor areas, which suggest different sets of information provided from separate portions of the cingulate cortex. A preliminary account of this study has appeared elsewhere (Wang et al., 1999).
2. Materials and methods Four Japanese monkeys (Macaca fuscata) weighing 5 – 7.5 kg were used in this study. The animals were cared for in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals, and Guidelines for Institutional Animal Care and Use published by our institute. After they were fully accustomed to sitting quietly in a primate chair with their limbs restrained during electrophysiolgical examination, they were anesthetized for surgery with ketamine hydrochloride (5 mg/kg, i.m.) and sodium pentobarbital (30 mg/kg, i.m.). Ketamine was added as needed. Under aseptic conditions, the skull was removed adequately to expose the MI, SMA, and preSMA. The dura mater was left intact. Acrylic recording chambers and head fixation bolts were implanted on the skull. After the surgery, monkeys were given antibiotics and returned to their cages. After a 1-week recovery period, electrophysiological examinations began. For intracortical microstimulation (ICMS) and for recording extracellular unit activity, we used glass-insulated Elgiloy microelectrodes with measured impedance of 0.8–1.0 MV (at 300 Hz). For stimulation, trains of 12 cathodal 200 ms pulses were applied at 333 Hz
through a constant current stimulator. Current intensities routinely ranged from 3 to 40 mA. However, 22 or 42 pulses and currents of up to 50 mA were also used for the pre-SMA and cingulate cortex, and for some parts of the SMA. Physiological examinations were performed in fully awake monkeys. ICMS was begun when the microelectrode reached a depth of 1.0 mm from the pial surface and, thereafter, was performed at every 500 mm of electrode advancement. In each area, we mapped the topographical organization based on ICMS effects, and on cell receptive fields of somatosensory responses. After identifying the forelimb areas of the SMA, preSMA, MI and cingulate cortex, electrolytic microlesions (anodal currents of 350 mC) were placed to use as reference points for later reconstruction of electrode tracks. Microlesions were made at the deepest points of microelectrode entry. Cortical depths at which ICMS effects were determined were calculated on the basis of depth reading by the stepping microdrive. Three different tracers were used: 5% diamidino yellow (DY, Sigma; dissolved in saline), 5% fast blue (FB, Sigma; dissolved in saline), and 5% wheat germ agglutinin conjugated to horseradish peroxidase (WGAHRP, Toyobo; dissolved in 0.5 M NaCl with 0.1 M mannose). On the basis of the map constructed by the electrophysiological studies, we selected the sites for tracer injections in the SMA, pre-SMA, and MI. The choice of tracers at each injection site and the amount injected in each animal are listed in Table 1. Tracers were pressure-injected at one or two depths with a 1.0-ml Hamilton syringe attached to the same manipulator as used for microelectrode insertion. The amount per injection was 0.3–0.4 ml for DY and FB, and 0.1–0.15 ml for WGA-HRP. The survival period was 3 weeks for fluorescent dyes (DY and FB) and 3 days for WGA-HRP. The survival period after each injection
Table 1 Injection site and tracers injecteda Monkey No.
Injection sites
Tracer
Number of injection sites
Total amount (ml)
1
MI SMA Pre-SMA
WGA-HRP DY FB
3 1 1
0.75 0.80 0.80
2
MI SMA Pre-SMA
WGA-HRP FB DY
3 1 1
0.75 0.80 0.80
3
MI Pre-SMA
WGA-HRP DY
3 1
1.20 0.80
4
MI SMA Pre-SMA
WGA-HRP DY FB
4 1 1
1.05 0.80 0.80
a Injection sites are abbreviated as MI, the forelimb area of the primary motor cortex; SMA, the forelimb area of the supplementary motor area; pre-SMA, the forelimb area of the presupplementary motor area.
Y. Wang et al. / Neuroscience Research 39 (2001) 39–49
41
Fig. 1. Photomicrographs showing injection sites (A, B and C) and labeled cells in the cingulate motor area (D and E). (A) diamidino yellow (DY) injection into the supplementary motor area (SMA). (B) Fast blue (FB) injection into the pre-supplementary motor area (pre-SMA). (C) Wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) injection into the primary motor cortex (MI). (D) FB- and DY-labeled cells under epifluorescent illumination. FB-labeled cells appear as blue spots. Arrows indicate DY-labeled cells. The nuclear localization of DY and the cytosolic localization of FB can be seen. (E) WGA-HRP-labeled cells under the mid-condition of brightfield and epifluorescent illumination in the same section. The dashed lines in D and E illustrate the borders of adjoining cortical layers (III, V, and VI). Calibration bars in A – C are 1 mm.
42
Y. Wang et al. / Neuroscience Research 39 (2001) 39–49
was found adequate for detecting corticocortical projection cells in M. fuscata. Fig. 1A – C shows photomicrographs of injection sites of fluorescent dyes and WGA-HRP. Under deep anesthesia with ketamine hydrochloride (5 mg/kg, i.m.) and sodium pentobarbital (50 mg/kg, i.m.), each monkey was perfused transcardially with 0.1 M phosphate buffer (pH 7.4) containing heparin. Fixative solutions were then perfused in the following order: 3% paraformaldehyde in 0.1 M phosphate buffer, 3% paraformaldehyde in 0.1 M phosphate buffer with 10% glycerin, 0.1 M phosphate buffer with 10% glycerin and 0.1 M phosphate buffer with 20% glycerin. The brains were removed, blocked, and for a few days before sectioning they were stored at 4°C in a solution containing 20% glycerin and 2% dimethyl sulfoxide in 0.1 M phosphate buffer (pH 7.4). Serial sections were made at 50 mm in the frontal plane on a freezing microtome. Each section was mounted on a gelatin-coated glass slide. Every alternate section was left unstained for examination with a fluorescence microscope, and was later Nissl-stained with cresyl violet for cytoarchitectonic examination. The remaining half of the sections was processed for HRP staining with tetramethylbenzidine. As shown in Fig. 1D and E, cells labeled with fluorescent dyes (DY and FB) can be clearly seen even in HRP-processed sections (Shindo et al., 1995). It was possible to examine cells labeled with WGA-HRP and with DY (or FB) in the single sections under both brightfield and epifluorescent illumination with 360-nm ultraviolet excitation. Thus, plotting was made in HRPprocessed sections using a computerized digital plotting system (MD2 Microscope Digitizer, Minnesota Digitizer, Minnesota Datametrics) to measure their locations in each section. In every fourth HRP-processed section, all labeled cells were plotted to examine the spatial distribution of projection cells in the cingulate cortex. WGA-HRP injection sites were considered to include the densely stained regions adjacent to the needle track and the surrounding halo. DY and FB injection sites were considered to include the first and second zones of Keizer et al. (1983). Each injection site was reconstructed from serial coronal sections with systematic measurements of its extent and spatial relation to the surrounding structures. To measure the density of projection cells, we performed a quantitative analysis of labeled cells. In each coronal section, the gray matter of the cortex in the mesial hemisphere above the corpus callosum was divided into unitary compartments (laminae of 500-mm width), perpendicular to the cortical surface. The number of labeled cells with each tracer was counted in each 500-mm lamina, using the computerized counting system.
3. Results
3.1. Physiological identification of injection sites The forelimb representation area of the SMA was identified by a combination of the following criteria: (1) ICMS evoked forelimb movements with thresholds of less than 30 mA; (2) cell activity related to the animal’s voluntary forelimb movements; (3) somatosensory responses evoked by touching the forelimb or manipulating forelimb joints; and (4) absence of visually evoked responses (Tanji and Kurata, 1979; Macpherson et al., 1982; Mitz and Wise, 1987; Tanji, 1994). A representative example of topographical mapping in the forelimb area of the SMA is shown in Fig. 2. Rostral to the forelimb area, the face was represented. Further rostrally, a separate forelimb area was found. The rostral area corresponded to the pre-SMA according to the following criteria: (1) ICMS-evoked complex forelimb
Fig. 2. Schematic diagram showing results of electrophysiological mapping and injection sites of tracers in the SMA and pre-SMA of monkey c 1. (Top) Top view of the left hemisphere showing the location of cortical injection sites (*). (Middle) Each label indicates a penetration site where movements were elicited by the intracortical microstimulation (ICMS) in the following body parts: Fa, face; El, elbow; Wr, wrist; D, digits; T, trunk; H, hindlimb. (Bottom) Based on histological examinations, representative electrode tracks at penetrations in and around injection sites are reconstructed (El1-El2, D3-El4), along with the directions indicated with interrupted lines. Evoked movements by the ICMS are shown at about 500-mm depths of the electrode tracks. DY and FB injections were made into the forelimb area of the SMA and pre-SMA, respectively. The spread of tracers is indicated by dark and light areas corresponding to the first and second zones of Keizer et al. (1983). Crosses denote injection points. CS, central sulcus; ARC, arcuate sulcus; PS, principle sulcus. Note that each tracer was confined within the forelimb representation area. A thin arrow indicates the caudal end of arcuate sulcus.
Y. Wang et al. / Neuroscience Research 39 (2001) 39–49
movements with higher threshold of 30 – 50 mA; (2) cell activity related to the animal’s voluntary forelimb movements; (3) little or no somatosensory responses to natural stimuli; and (4) presence of visual responses to flash of lights or moving objects (Luppino et al., 1991; Matsuzaka et al., 1992). FB or DY was injected into the central focus of the forelimb area of the SMA or pre-SMA at depths of 1.0 – 4.0 mm from the pial surface. The spread of injections was no larger than 2 mm in diameter, and was confined to the forelimb representation within the gray matter of the SMA and preSMA. The forelimb areas of the MI were identified by the ICMS and somatosensory responses (Sessle and Wiesendanger, 1982; Sato and Tanji, 1989; Huntley and Jones, 1991). We surveyed extensively the entire MI, both the rostral bank of the central sulcus and the surface of the precentral gyrus. A representative example of topographical mapping in the MI observed in monkey c1 is shown in Fig. 3. Multiple injections were made into the forelimb area of the MI. Injections were placed at depths of 1 – 1.5 and 2 – 2.5 mm from the pial surface in the rostral bank of the central sulcus, and at depths of 1–1.5 mm from the pial surface in the cranial part of the precentral gyrus. The spread of
Fig. 3. Schematic diagram showing results of electrophysiological mapping and injection sites of a tracer in the MI of monkey c1. (Top-right) top view of the left hemisphere showing the location of cortical injection sites (asterisks). (Bottom) precentral sites of microelectrode entry. Each label indicates a penetration site where movements were elicited from more than a half of the ICMS delivery in the following body parts: Fa, face; Ne, neck; D, digits; Wr, wrist; El, elbow; Sh, shoulder. Based on histological examinations, representative electrode tracks at penetrations around injection sites are reconstructed (El1-Wr2, El3-Wr4, D5-D6), along with the directions indicated with interrupted lines and drawn. Evoked movements by the ICMS are shown at about 500-mm depth of the electrode tracks. Three WGA-HRP injections were made into the forelimb area of the MI. The spread of tracers is indicated by shaded areas with dark and light areas. Crosses denote injection points. CS, central sulcus; ARC, arcuate sulcus; PS, principle sulcus.
43
injections was within 2.5 mm in diameter, and confined to each representation within the gray matter of the MI. Later histological analysis showed that injections were localized within cytoarchitectonic area 4 of Brodmann characterized by the presence of giant pyramidal cells in layer V. Frontal reconstructions through the three injection sites are illustrated in Fig. 3.
3.2. Distribution of retrogradely labeled cingulate cells Three different tracers were injected into the forelimb area of the MI, SMA and pre-SMA of three monkeys and two different tracers were injected into the forelimb area of the MI and pre-SMA in one monkey (Table 1). Among them, one monkey was used for the ICMS mapping of the cingulate motor areas as well as the SMA and pre-SMA. Fig. 4 shows the distribution of labeled cells in an unfolded map of the medial surface of the hemisphere, and the topographical motor map obtained by the ICMS (monkey c4). In this monkey, WGA-HRP was injected into the MI, and DY and FB were injected into the SMA and pre-SMA, respectively (Table 1). As shown in Fig. 4, two clusters of WGA-HRP labeled cells were observed in the cingulate cortex rostrocaudally in the banks of the cingulate sulcus. The large caudal cluster composed of densely packed labeled cells was located at the dorsal and ventral banks of the cingulate sulcus in a region termed areas 6c and 23c by Dum and Strick (1991), LC by Von Bonin and Bailey (1947), and area 23c by Vogt et al. (1987). The rostral cluster of labeled cells was located at the rostral portion of cingulate cortex corresponding to FDL by Von Bonin and Bailey (1947), and area 24c by Vogt et al. (1987). This cluster was found in the ventral and dorsal banks of the cingulate sulcus at the level of the pre-SMA and SMA. In this report, we called the rostral and caudal foci of MI-projection cells as the CMAr and CMAc. A cluster of HRP-labeled cells was also found in the SMA, but not in the pre-SMA. In the monkey c 4, DY-labeled cells projecting to the SMA were widely distributed over the medial wall of the hemisphere, with several clusters. Some clusters of DY-labeled cells overlapped with the CMAr (Fig. 5C) and CMAc (Fig. 5D). The overlap was particularly extensive in the CMAc. In contrast, FB-labeled cells projecting to the pre-SMA were distributed in the rostral part of the cingulate cortex and in the superior frontal gyrus rostral to the injection site. Although FB-labeled cells showed partial overlap with the rostral part of the CMAr, few FB-labeled cells were observed in and around the CMAc. Fig. 4C shows the topographic motor map obtained by the ICMS in the same monkey. Generally, the areas where forelimb movements were evoked corresponded to regions in which cells project to the forelimb areas of
44
Y. Wang et al. / Neuroscience Research 39 (2001) 39–49
Fig. 4. Plots of three different retrogradely labeled cells on the medial wall of hemisphere in monkey c4 (B) and the topographical motor map in the same region by the ICMS (C). (A) Top view of the left hemisphere showing the location of cortical injection sites (asterisks). CS, central sulcus; ARC, arcuate sulcus; PS, principle sulcus; Cing. S, cingulate sulcus; CC, corpus callosum. The letters a, b, c, and d in the schematic section drawing inset at top correspond to a, b, c, and d in B and C. Arrows in B and C indicate the caudal end of arcuate sulcus. In B, each dot represents two to five labeled cells.
Y. Wang et al. / Neuroscience Research 39 (2001) 39–49
45
Fig. 5. Distribution of retrogradely labeled cells in monkey c1. Each dot represents two to five labeled cells. (Top) top view of the left hemisphere showing the location of cortical injection sites (asterisks). The approximate positions of the coronal sections are specified by lines labeled with A to D. (Middle and Bottom) Coronal sections are arranged from A to D. Green, FB-labeled cells; blue, DY-labeled cells; red, WGA-HRP-labeled cells.
the MI, namely CMAr and CMAc. The hindlimb movements were elicited in the areas located caudal to the CMAc and in a region between the CMAr and CMAc. Fig. 5 shows the distribution of labeled cells in coronal sections obtained at four rostro-caudal levels (monkey c1). WGA-HRP-labeled and DY-labeled cells were overlapping extensively in the banks of the cingulate sulcus, both in the CMAr (Fig. 5C) and CMAc (Fig. 5D). However, in overlapping regions, the HRP- and DY-labeled cells were interdigitated, rather
than intermixed. The number of double-labeled cells was less than 2% of labeled cells in the CMAr and CMAc. As shown in Fig. 5A and B, FB-labeled cells were concentrated around the cingulate sulcus of the anterior cingulate cortex.
3.3. Quantitati6e analysis of distribution of projection cells To determine more precisely the spatial distribution and density of projection cells to the MI, SMA, and
46
Y. Wang et al. / Neuroscience Research 39 (2001) 39–49
pre-SMA, quantitative analysis was made on the basis of data obtained in three monkeys. As mentioned in Section 2, the cingulate cortex in each section was divided into unitary compartments of 500-mm laminae perpendicular to the cortical surface. In each lamina, the numbers of labeled cells with the three tracers were counted and plotted on the unfolded medial surface map of the hemisphere. The density map obtained from the data in monkey c 1 is shown in Fig. 6. Two clusters of labeled cells projecting to the forelimb area of the MI were observed rostocaudally along the cingulate sulcus, as in Fig. 4B. The areal size of the CMAc was larger than that of the CMAr. The density of WGA-HRP labeled cells in the CMAc (8.89: mean number of labeled cells/lamina) was
significantly higher (P B 0.001, t-test) than that in the CMAc (4.74/lamina). Labeled cells projecting to the forelimb area of the SMA were widely distributed in the cingulate cortex. However, high-density regions were found in the CMAr and CMAc and their adjacent regions. The mean density of labeled cells projecting to the SMA was greater (PB 0.001, t-test) in the CMAr (17.61/lamina) than in the CMAc (11.07/lamina). Projection cells to the preSMA were mainly located in the rostral part of the cingulate cortex and in a small part of the cingulate gyrus. These regions showed partial overlap with the region projecting to the SMA and ventral part of the CMAr, but showed little overlap with the CMAc.
Fig. 6. Quantified plots of three different retrogradely labeled cells on the medial wall of hemisphere in monkey c1. (Top) top view of the left hemisphere showing the location of cortical injection sites (*). In this figure and in Fig. 6, red, blue and green bars indicate projection cells to the MI, SMA and pre-SMA, respectively. Sizes of bars indicate the density of labeled cells in each bin (see Section 2).
Y. Wang et al. / Neuroscience Research 39 (2001) 39–49
47
4. Discussion
Fig. 7. Frequencies of occurrences of unitary compartments (laminae) within the CMAr (left) and CMAc (right), possessing labeled cells projecting to the SMA and/or pre-SMA, in addition to cells projecting to the MI. The data obtained from three monkeys (c 1, 2 and 4) were averaged and expressed as percentages.
The quantitative analysis was performed on the data obtained from three monkeys, giving rise to basically the same results as in monkey c1. To summarize the degree of overlap of projections to the three areas, we first analyzed how many of the unitary compartments (individual laminae) in the CMAr and CMAc contained labeled cells projecting to the SMA and/or preSMA, in addition to projection cells to the MI. For this purpose, we calculated the average number of compartments including labeled cells in various combinations, from the data for the three monkeys. Fig. 7 shows the frequency of occurrences of the compartments, expressed as percentages. In both the CMAr and CMAc, a majority of the compartments (57 and 71%, respectively) projects to the SMA, as well as to the MI. In the CMAr, 19% of the compartments included cells projecting to all three areas. In contrast, in the CMAc, only a small minority of compartments possessed cells projecting to the pre-SMA. Subsequently, we examined the degree of overlap within the areas projecting to the SMA and to the pre-SMA, as shown in Fig. 8. Thirty four percent of the pre-SMA-projecting compartments included SMA-projecting cells, whereas 16% of the SMA-projecting compartments included pre-SMA-projecting cells. The compartments with projection cells to the pre-SMA and MI were small in number, compared with compartments with shared projections to the SMA and MI. The great majority of pre-SMA-projecting compartments (85%) did not include MI-projection cells.
In this study we investigated the spatial distribution of cingulate cells projecting to the forelimb areas in the MI, SMA, and pre-SMA, with a technique of injecting three different tracers into physiologically identified motor areas. We found that both the CMAr and CMAc, defined as cingulate motor areas projecting to the MI, have extensive projections to the SMA. In contrast, only a small fraction of cingulate motor areas contained cells projecting to the pre-SMA. Especially, the projection from the CMAc to the pre-SMA was meager. The cingulate areas projecting to the pre-SMA and SMA exhibited overlap in 34% of pre-SMA-projecting territory and in 16% of SMA-projecting territory. On the other hand, about a half of the SMA-projecting area, and also a half of the pre-SMA projecting area included no cells projecting to other motor areas. These findings indicate that the MI and SMA receive common sources of information from the cingulate motor areas, whereas the cingulate inputs for the MI and pre-SMA are largely separate. Furthermore, the input sources for the pre-SMA and SMA arise primarily from the rostral and caudal parts of the cingulate motor areas, with a partial overlap. It seems important, however, to acknowledge limitations in interpreting our present data, although the forelimb representation areas were physiologically identified and tracer injections were made in a portion of each area. We have to be cautious about possible underestimates of the number and distribution of projection cells. It should also be mentioned that the tracer injections into hindlimb or facial representation areas are likely to reveal different sets of projection cells (Morecraft and Van Hoesen, 1993; Morecraft et al., 1996; Tokuno et al., 1997).
Fig. 8. Frequencies of occurrences of unitary compartments projecting to the pre-SMA (left) and SMA (right), possessing a variety of combinations of cells projecting to other three motor areas.
48
Y. Wang et al. / Neuroscience Research 39 (2001) 39–49
The present study confirmed previous reports (Luppino et al., 1993; Morecraft and Van Hoesen, 1993; He et al., 1995;) in that the density of projection cells to the MI is larger in the CMAc than in the CMAr. We further found in this study that the CMAr and its adjacent regions densely project to the SMA. Further rostrally, the banks of the cingulate sulcus project to the pre-SMA without much projection to other motor areas. These findings suggest that the rostral part of the cingulate cortex is heterogeneous with respect to efferent projections, while the caudal part appears more homogeneous with prominent outputs to the MI and SMA. A previous study from our laboratory (Shima et al., 1991) found that the cell activity in the caudal cingulate motor area showed resemblance to that in the MI, primarily in relation to execution of movements. On the other hand, cells in the rostral cingulate motor area exhibited relations to motor preparation (Shima et al., 1991) or to reward-based motor selection (Shima and Tanji, 1998). Very recently, anterior cingulate cells were found to be active preferentially in relation to routine or non-routine sequential motor behavior (Procyk et al., 2000). These single-cell recording studies imply the role of rostral cingulate cortex in cognitive aspects of motor behavior. This view finds support from a large number of brain imaging studies in human subjects (Carter et al., 1998; Petit et al., 1998; Kiehl et al., 2000; for review, see Picard and Strick, 1996). Considering the diversity of outputs from the rostral cingulate cortex as found in this study, it is necessary in future studies to determine whether various aspects of cell activity are specialized in certain areas with specific output projections.
Acknowledgements We thank M. Kurama and Y. Takahashi for technical assistance. The work was supported by grants from the Ministry of Education, Science, and Culture of Japan (08 279 101 and 09 308 032), and by Japan Science and Technology Corporation.
References Carter, C.S., Braver, T.S., Barch, D.M., Botvinick, M.M., Noll, D., Cohen, J.D., 1998. Anterior cingulate cortex, error detection, and the online monitoring of performance. Science 280, 747–749. Dum, R.P., Strick, P.L., 1991. The origin of corticospinal projections from the premotor areas in the frontal lobe. J. Neurosci. 11, 667 – 689. He, S.Q., Dum, R.P., Strick, P.L., 1995. Topographic organization of corticospinal projections from the frontal lobe: motor areas on the medial surface of the hemisphere. J. Neurosci. 15, 3284 – 3306.
Huntley, G.W., Jones, E.G., 1991. Relationship of intrinsic connections to forelimb movement representations in monkey motor cortex: a correlative anatomical and physiological study. J. Neurophysiol. 66, 390 – 413. Keizer, K., Kuypers, H.G.J.M., Huisman, A.M., Dann, O., 1983. Diamidino yellow dihydrochloride (DY:2HCl): a fluorescent retrograde neuronal tracer which migrates only very slowly out of the cell and can be used in combination with TB and FB in double labeling experiments. Exp. Brain Res. 51, 179 – 191. Kiehl, K.A., Liddle, P.F., Hopfinger, J.B., 2000. Error processing and the rostral anterior cingulate: an event-related fMRI study. Psychophysiology 37, 216 – 223. Luppino, G., Matelli, M., Camarda, R.M., Gallese, V., Rizzolatti, G., 1991. Multiple representations of body movements in mesial area 6 and the adjacent cingulate cortex: an intracortical microstimulation study in the macaque monkey. J. Comp. Neurol. 311, 463 – 482. Luppino, G., Matelli, M., Camarda, R., Rizzolatti, G., 1993. Corticocortical connections of area F3 (SMA-proper) and area F6 (pre-SMA) in the macaque monkey. J. Comp. Neurol. 338, 114– 140. Macpherson, J.M., Marangoz, C., Miles, T.S., Wiesendanger, M., 1982. Microstimulation of the supplementary motor area (SMA) in the awake monkey. Exp. Brain Res. 45, 410 – 416. Matsuzaka, Y., Aizawa, H., Tanji, J., 1992. A motor area rostral to the supplementary motor area (presupplementary motor area) in the monkey: neuronal activity during a learned motor task. J. Neurophysiol. 68, 653 – 662. Mitz, A.R., Wise, S.P., 1987. The somatotopic organization of the supplementary motor area: intracortical microstimulation mapping. J. Neurosci. 7, 1010 – 1021. Morecraft, R.J., Van Hoesen, G.W., 1992. Cingulate input to the primary and supplementary motor cortices in the rhesus monkey: evidence for somatotopy in areas 24c and 23c. J. Comp. Neurol. 322, 471 – 489. Morecraft, R.J., Van Hoesen, G.W., 1993. Frontal granular cortex input to the cingulate (M3), supplementary (M2) and primary (MI) motor cortices in the rhesus monkey. J. Comp. Neurol. 337, 669 – 689. Morecraft, R.J., Schroeder, C.M., Keifer, J., 1996. Organization of face representation in the cingulate cortex of the rhesus monkey. Neuroreport 7, 1343 – 1348. Muakkassa, K.F., Strick, P.L., 1979. Frontal lobe inputs to primate motor cortex: evidence for four somatotopically organized ’premotor’ areas. Brain Res. 177, 176 – 182. Petit, L., Courtney, S.M., Ungerleider, L.G., Haxby, J.V., 1998. Sustained activity in the medial wall during working memory delays. J. Neurosci. 18, 9429 – 9437. Picard, N., Strick, P.L., 1996. Motor areas of the medial wall: a review of their location and functional activation. Cereb. Cortex 6, 342 – 353. Procyk, E., Tanaka, Y.L., Joseph, J.P., 2000. Anterior cingulate activity during routine and non-routine sequential behaviors in macaques. Nat. Neurosci. 3, 502 – 508. Sato, K.C., Tanji, J., 1989. Digit-muscle responses evoked from multiple intracortical foci in monkey precentral motor cortex. J. Neurophysiol. 62, 959 – 970. Sessle, B.J., Wiesendanger, M., 1982. Structural and functional definition of the motor cortex in the monkey (Macaca fascicularis). J. Physiol. (London) 323, 245 – 265. Shima, K., Tanji, J., 1998. Role for cingulate motor area cells in voluntary movement selection based on reward. Science 282, 1335 – 1338. Shima, K., Aya, K., Mushiake, H., Inase, M., Aizawa, H., Tanji, J., 1991. Two movement-related foci in the primate cingulate cortex observed in signal-triggered and self-paced forelimb movements. J. Neurophysiol. 65, 188 – 202.
Y. Wang et al. / Neuroscience Research 39 (2001) 39–49 Shindo, K., Shima, K., Tanji, J., 1995. Spatial distribution of thalamic projections to the supplementary motor area and the primary motor cortex: a retrograde multiple labeling study in the macaque monkey. J. Comp. Neurol. 357, 98–116. Tanji, J., Kurata, K., 1979. Neuronal activity in the cortical supplementary motor area related with distal and proximal forelimb movements. Neurosci. Lett. 12, 201–206. Tanji, J., 1994. The supplementary motor area in the cerebral cortex. Neurosci. Res. 19, 251–268. Tokuno, H., Takada, M., Nambu, A., Inase, M., 1997. Reevaluation of ipsilateral corticocortical inputs to the orofacial region of the primary motor cortex in the macaque monkey. J. Comp. Neurol.
.
49
389, 34 – 48. Vogt, B.A., Pandya, D.N., Rosene, D.L., 1987. Cingulate cortex of the rhesus monkey: I. Cytoarchitecture and thalamic afferents. J. Comp. Neurol. 262, 256 – 270. Von Bonin, G., Bailey, P., 1947. The neocortex of Macaca mulatta. In: Monographs in Medical Sciences, vol. 5. University of Illinois Press, Urbana, IL, p. 136. Wang, Y., Shima, K., Sawamura, H., Tanji, J., 1999. A multiple labeling study of projection neurons in the cingulate cortex to the primary motor cortex, the supplementary motor area, and the pre-supplementary motor area. Neurosci. Res. 23, S199.