Axon trajectories in local circuits of the primary motor cortex in the macaque monkey (Macacafuscata)

Neuroscience Research 39 (2001) 233– 245 www.elsevier.com/locate/neures

Axon trajectories in local circuits of the primary motor cortex in the macaque monkey (Macaca fuscata) Akiko Yamashita, Tomio Arikuni * Department of Anatomy, Nihon Uni6ersity School of Medicine, 30 -1 Oyaguchi-Kamimachi, Itabashi-ku, Tokyo 173 -0032, Japan Received 24 May 2000; accepted 20 October 2000

Abstract The intrinsic trajectories and terminal arbors of two axons and one horizontal axon collateral within the primary motor cortex (M1) were studied in the macaque monkey using injections of biotinylated dextran amine (BDA) into the putative primary forelimb motor cortex, and two-dimensional (2-D) reconstruction of the individually labeled axons and collateral. (1) A long collateral of the main axon from a large pyramidal cell in layer Vb of the putative forelimb area on the anterior bank of the central sulcus coursed horizontally anteriorly for 3 mm and formed a terminal arbor in layer III of M1. (2) The main axon of a pyramidal cell in layer IIIa +b of the putative forelimb area on the precentral gyrus descended into the white matter and then entered the anterior bank of the central sulcus to form a terminal arbor in layers III and V. (3) The main axon of a pyramidal cell in layer IIIc of the putative forelimb area on the precentral gyrus descended and bifurcated in the white matter. One branch entered the anterior bank of the central sulcus to form a terminal field in layer VI. These results indicate that some local axons and horizontal axon collaterals arising from M1 reach their single targets within M1 to form single terminal fields. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Horizontal connection; Axon collateral; M1; Precentral gyrus; BDA (biotinylated dextran amine)

1. Introduction The primary motor cortex (M1) has been studied mainly in relation to control of single muscles, muscle groups, and movements of body parts. Earlier mapping experiments used electrical stimulation of the cortical surface and indicated a single somatotopic representation of the major subdivisions of the body (Humphrey, 1986, for review); the face, forelimb, trunk, and hindlimb of the body are represented in separate cortical regions on the precentral gyrus. More recent studies employed intracortical microstimulation (ICMS), single unit recordings, spike triggered averaging of muscle activities, and/or the electromyography in monkeys or the functional magnetic resonance imaging and the positron emission tomography in humans (Asanuma and Rosen, 1972; Kwan et al., 1978; Murphy et al., 1978; Strick and Preston, 1978; Wong et al., 1978; * Corresponding author. Tel: + 81-3-3972-8111; fax: +81-3-39728292. E-mail address: [email protected] (T. Arikuni).

McGuinness et al., 1980; Humphrey and Reed, 1983; Buys et al., 1986; Lemon et al., 1987; Huang et al., 1988; Sato and Tanji, 1989; Donoghue et al., 1992; Inase and Tanji, 1995; Kawashima et al., 1995; Sanes et al., 1995; Shindo et al., 1995; Baker et al., 1997). These studies show that same muscles and joints or body parts are represented in several spatially separate sites in the representation zone of each of the major subdivisions of the body on the precentral gyrus. There is still controversy, however, regarding the size, pattern, and location of motor representation (Schieber and Hibbard, 1993). Anatomic tracing studies demonstrate that there is a profuse set of intrinsic connections within the major representation areas, but that the face, forelimb, and hindlimb areas are not interconnected (Ku¨nzle, 1976; Goldman and Nauta, 1977; Jones et al., 1978; Godschalk et al., 1984; Matelli et al., 1984; Tokuno et al., 1997). Huntley and Jones (1991) used ICMS to map the forelimb representation in the monkey and then injected horseradish peroxidase (HRP) into the ICMSdefined sites. They report that a single representation

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site in the hand area connected reciprocally with a variety of representation sites across the forelimb area. Similar intra-areal connections were observed in the forelimb area of M1 in the cat (Keller and Asanuma, 1993; Ghosh, 1997). Such intra-areal connectivity is thought to arise from the pyramidal cells of M1 that emit long, horizontal or vertical axon collaterals (Landry et al., 1980; DeFelipe et al., 1986; Ghosh and Porter, 1988; Ghosh et al., 1988). On the other hand, experiments with brain slice preparations have demonstrated that the horizontal and vertical (ascending) connections in M1 participate in producing long-term potentiation (Aroniadou and Keller, 1995; Hess et al., 1996) and excitatory synaptic responses (Keller, 1993). The purpose of the present study is to determine whether or not a single axon originated from M1 projects to multiple sites in the same M1. To address this issue, a tracer biotinylated dextran amine (BDA) was chosen because it clearly labels axon terminals with terminal boutons discernible under a light microscope. BDA was injected into the putative forelimb region of M1 of the macaque monkeys and the intracortical trajectories of axons or axon collaterals arising from pyramidal cells of M1 were investigated. Preliminary results of the present study have been published earlier (Arikuni and Yamashita, 1997).

2. Materials and methods

2.1. Animal preparation Two Japanese monkeys (Macaca fuscata; 2.5 – 4.0 kg; cases 96129 and 9657R) were used. The animals were pretreated with ketamine hydrochloride (10 mg/kg, i.m.) and then anaesthetised with pentobarbital sodium (35 mg/kg) through a catheter attached to the saphenous vein. During surgery and BDA injection, an electrolyte solution containing glucose and pentobarbital sodium was given through the catheter when necessary to maintain a stable condition and an adequate level of anesthesia. The monkeys were kept on a heating pad to maintain body temperature and mounted in a stereotaxic apparatus. Immediately before surgery, Mannitol solution (15 w/v%, 60 – 80 ml) was given through the catheter to reduce brain volume. All surgical procedures were performed aseptically. The experimental protocol was approved by the Animal Research Committee of Nihon University School of Medicine, and was in accordance with the Guide for the Care and Use of Laboratory Animals established by NIH and the Guide for the Care and Use of Laboratory Primates established by the Primate Research Institute, Kyoto University.

2.2. BDA injection Craniotomy was performed over the precentral gyrus. The dura mater over M1 was cut and reflected. The primary forelimb motor cortex was identified by its gross topographic location on the precentral gyrus in reference to the published maps of face and forelimb representations on M1 in the macaque brain (Jankowska et al., 1975; Kwan et al., 1978; Huang et al., 1988; Huntley and Jones, 1991; Tokuno and Tanji, 1993; Inase and Tanji, 1995; Shindo et al., 1995; Armand et al., 1997). In addition, in the preliminary experiments, microelectrodes were introduced to stimulate the forelimb and face regions on the precentral gyrus to enable us to locate the primary forelimb area in two Japanese monkeys. The boundary between the oro-face and forelimb representations on the precentral gyrus was 17 and 18 mm lateral to the medial margin of the hemisphere, respectively. In another preliminary study, a small dose of GABAA antagonist, bicuculline methioide (Sigma-Aldrich, Japan) was injected into a site of M1 on the precentral cortex, lying posterior to the spur of the arcuate sulcus. After the bicuculline injection, twitches of the digit muscles occurred on the contralateral side, indicating that the site is the finger area. Thus, although the motor maps vary among monkeys, M1 along the anterior curved portion of the central sulcus (the genu) can be presumed as the region of the primary forelimb motor area, referred to as the putative forelimb motor area in this study. A micropipette with a tip diameter of 15 mm, filled with 5% BDA (10 000 MW; Molecular Prove Inc., Eugene, OR) in 0.9% saline, was inserted into the putative forelimb area of M1 at a depth of 1.0 to 2.5 mm from the cortical surface (Fig. 1A and B). BDA was injected by iontophoresis (positive current pulses of 0.9 –1.0 mA, 500 ms duration, half-duty cycle for 20 min). After injection of BDA, the dura mater was sutured, and the removed bone flap was glued to the skull. The animals were given an antibiotic and removed from the stereotaxic frame. They were returned to their cages and provided with water, soft animal pellets and fruit.

2.3. Tissue preparation After a survival period of 48 h, the animals were pretreated with ketamine hydrochloride (10 mg/kg, i.m.) and then euthanised by an overdose of pentobarbital sodium. They were perfused through the ascending aorta with 300 ml of 0.02% heparin sodium (1000 U/ml) in ice-cold 0.15 M NaCl or Ringer’s solution, followed by 3000 ml of 2% paraformaldehyde and 0.5% glutaraldehyde in ice-cold 0.1 M phosphate buffer (PB), pH 7.4. The brains were immediately removed from the skull and photographs were taken of the external surfaces of each brain, after which they were dissected into

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blocks and postfixed overnight in 4% paraformaldehyde in PB at 4°C. The brain blocks were immersed in 30% sucrose in PB at 4°C until they sank. Frontal or parasagittal 50 mm thick sections were cut on a freezing microtome. Every free-floating section was pretreated overnight with 0.7% Triton-X, 0.1% bovine serum albumin (BSA) and 0.02% sodium azide in phosphate buffered saline (PBS) in a refrigerator. BDA was then visualised with the avidin-biotin-complex peroxidase method using the ABC elite kit (Vector Laboratories, Burlingame, CA). The sections were reacted with ABC solution for 2 h following incubation in 20 mg/ml diaminobenzidine tetrahydrochloride (DAB) and 0.05% nickel ammonium sulfate in 0.05 M Tris – HCl buffer, pH 7.6 for 2 h at 4°C in a refrigerator. They were then reacted with 0.3% H2O2, 20 mg/ml DAB, and 0.05% nickel ammonium sulfate in 0.05 M Tris – HCl buffer for several minutes. The sections were mounted on gelatin-coated slides, dehydrated, cleared in xylene, and covered with coverslips.

2.4. Reconstruction, analysis and photomicrographs Cell somata, dendrites, axons and terminal arbors were reconstructed from serial frontal or parasagittal

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sections with a camera lucida attached to a light microscope (Olympus, BX50) at × 40, × 100, × 200, and × 1000 magnification. First, individual cells and axons were drawn in every section using a camera lucida and X4, X10, X20 objectives to provide overall orientation. Next, these cells and axons were drawn in every section using a camera lucida and a X100 oil immersion objective, and 2-D reconstructions were prepared. Blood vessels and other profiles in the vicinity of the selected cells or axons were used to match corresponding points in the same or different sections. The 2-D reconstructions were scanned into a computer (Power Macintosh 7600/132). The scanned images were unified on the same computer to provide whole images of 2-D reconstruction of the soma, axons, and terminal arbors in appropriate sizes, using Adobe Illustrator (v5.5) software (Adobe Systems Incorporated). In these two-dimensional reconstructions the continuous axon branch at the crossing points of branches was displayed throughout in the same color. The border between the white and the gray matters, and the borders of cortical layers were identified by using non-specific background staining of neuropil and cell somata, especially light stained pyramidal cells in layers IIIc or Vb (Fig. 1C and D; Fig. 2E and F). Non-specific staining could not be detected in the apical

Fig. 1. Photographs of the dorsal view of Japanese monkey brains showing points of micropipette insertion into the precentral gyrus and approximate location of targets of local axons and a horizontal axon collateral, and photomicrographs showing sites of BDA injection. (A) Case 96129. A micropipette was inserted into the putative forelimb motor cortex at a point (black spot) on the crown of the anterior bank of the central sulcus on the left hemisphere. A hatched rectangle depicts approximate location of the terminal field formed by a long horizontal collateral of a large pyramidal cell in layer Vb. The number 3 next to the hatched rectangle indicates that the terminal field is shown in Fig. 3. (B) Case 9657R. A micropipette was inserted into the putative forelimb motor cortex at a point (black spot) on the precentral gyrus 4 mm anterior to the central sulcus on the right hemisphere. Hatched rectangles depict approximate locations of both a terminal field of axon arising from a layer IIIa +b pyramidal cell and a terminal field formed by axon branch of a layer IIIc pyramidal cell. The numbers 4 and 5 next to the rectangles indicate that the terminal fields are shown in Fig. 4 and Fig. 5, respectively. (C) A low-power photomicrograph showing the site of BDA injection (black reaction products of BDA) in the anterior bank of the central sulcus of case 96129. The site of injection was circumscribed in layers V and VI. (D) A low-power photomicrograph showing the site of BDA injection in the precentral gyrus of case 9657R. The site of injection was restricted to layers II, III, and V. AS, arcuate sulcus; CS, central sulcus; Fa, face motor cortex; Fl, forelimb motor cortex; Hl, hindlimb motor cortex; PS, principal sulcus; S, spur of arcuate sulcus; Vb, layer Vb. Scale bars in A and B, 5 mm, and in C and D, 500 mm.

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Fig. 2. Composite photographs and photomicrographs showing terminal arbors, axon varicosities, spine-like protrusions, terminal boutons, and BDA-labeled cells. (A) Composite photomicrograph of terminal arbors in layer III provided by a long horizontal axon collateral arising from a large pyramidal cell in layer Vb of case 96129 (see also Fig. 3B). BDA-labeled terminal boutons surround the soma of two unlabeled cells (asterisks). (B) Composite photomicrograph of terminal arbors in layer III provided by the main axon of a pyramidal cell in layer IIIa + b of case 9657R (see also Fig. 4A). (C) Composite photomicrograph of terminal arbors in layer VI provided by axon branch of a pyramidal cell in layer IIIc of case 9657R (see also Fig. 5). In A, B and C, a number of axon varicosities (boutons en passant, arrows), spine-like protrusions (double arrowheads), and terminal boutons (arrowheads) are easily discerned. (D) High-power photomicrograph showing a BDA-labeled axon terminal from the same section as in C with terminal boutons. (E) Low-power photomicrograph showing a BDA-labeled pyramidal cell in layer IIIa +b that provided a terminal field in layers III and V in the anterior bank of the central sulcus of case 9657R (see also Fig. 4). (F) Low-power photomicrograph showing a BDA-labeled pyramidal cell (large arrowhead) in layer IIIc in the vicinity of the site of injection that provided a terminal field in layer VI of case 9657R (see also Fig. 5). Small arrowheads point to somata of non-specifically stained large pyramidal cells in layer Vb. The Roman numerals denote cortical layers. Scale bars in A, B, C and E, 50 mm; in D, 10 mm; in F, 100 mm.

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and basal dendrites of the pyramidal cells. The non-specific background staining was adjusted by changing reaction time of the last step for the DAB/nickel reaction. In addition, after tracing the axon trajectories, several selected sections with axon arbors (Figs. 2, 4 and 5) and the injection sites (Fig. 1) were counterstained for Nissl with neutral red to identify cytoarchitectonic areas and cortical layers. Using these counter-stained sections, we examined the locations of pyramidal cells issuing the local axons and axon arbors observed in this study. Cortical layers of M1 were delineated in accordance with Bonin and Bailey (1947) and Braak (1984). Terminal fields and axon arbors were defined as the portion of axon consisting of multiple axon branches with terminals and varicosities. Some axon arbors and total terminal fields were measured along the major axis of the reconstructed arbor and the terminal field. Others were calculated by multiplying sections with arbors or terminal field by 50 mm. The distance of axon trajectories was measured along the reconstructed axons. The size of the soma was measured on the reconstructed pyramidal cells. To visualise the full length of axons and of the axon collateral with their terminal-like structures, photomicrographs of Fig. 2A, B and C were taken with MCID, an image analysis computer system (MCID Co. Ltd., Canada). The light microscope (Olympus, BX50) was controlled by the MCID system, and photomicrographs of BDA-labeled structures at every 2 mm-depth step in a single section taken with a digital camera were fed into the MCID. The MCID automatically made a composite photomicrograph depicting a whole image of the axon and its branches with terminal specialisation within each single 50 mm-thick section.

3. Results

3.1. Injection sites Injection of BDA was made into the putative forelimb motor cortex on the precentral gyrus, which cytoarchitectonically corresponds to area 4b (Vogt and Vogt, 1919; Watanabe-Sawaguchi et al., 1991). Injection sites were qualitatively defined as including the central zone of densely dark reaction products and the peripheral zone where faint reaction products and heavily labeled cells and fibres were present. The injection

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site in case 96129 was circumscribed in layers V and VI of the middle one-third of the anterior bank of the central sulcus at 14 mm lateral to the medial margin of the left hemisphere (Fig. 1A and C). The injection site in case 9657R was circumscribed in layers II, III and V of the putative forelimb motor cortex at 4 mm anterior to the central sulcus and 13 mm lateral to the medial margin of the right hemisphere (Fig. 1B and D). These injection sites presumably correspond to representation of the forelimb, judging from the motor maps of others’ earlier studies (Huntley and Jones, 1991; Inase and Tanji, 1995; Shindo et al., 1995). Consistent with previous studies in which Biocytin was used (Clarke et al., 1993; Aumann et al., 1998), BDA injection produced a number of anterogradely labeled axons and a small number of retrogradely labeled cells around the injection sites (Fig. 2F). With regard to labelling of intracortical axons, myelinated thick axons whose nodes of Ranvier sometimes gave rise to collaterals, thin axons with collaterals, and axon varicosities (boutons en passant) and terminals with boutons were clearly labeled (Fig. 2A–D). Hence, axon and axonal branches with terminals were distinguished easily from passing fibres.

3.2. A long horizontal collateral of axon of a large pyramidal cell in layer Vb In case 96129, a large pyramidal cell in layer Vb was labeled 400 mm posterior, inferior, and medial away from the injection site in M1 on the anterior bank of the central sulcus (Fig. 3A). This pyramidal cell had a large cell body (small and large diameters, 20×33 mm) and a thick apical dendritic trunk whose labelling was lost in the middle of layer III. Thick basal dendrites radiated from the soma, but they were insufficiently labeled. A main axon emerged from the base of the soma and descended into the white matter. The main axon was covered by a myelin sheath 37 mm distant from the soma because it increased in diameter, and it issued a thick collateral of the same diameter as the main axon below the soma, suggesting a myelinated branch and, then, seven fine collaterals in layer Vb. These fine collaterals ramified in layer Vb in the right lower quadrant to the soma and extended into the upper part of layer VI. A terminal field formed by these fine collaterals spanned 292 mm mediolaterally and 182 mm dorsoventrally. In the present study, the main axon of this pyramidal cell was not traced further. In the

Fig. 3. Trajectory and terminal arbors of the long horizontal axon collateral from a large pyramidal cell in layer Vb of case 96129. (A) Brain diagram shows location of the large pyramidal cell of origin in layer Vb (red triangle) in the anterior bank of the central sulcus, pointing out the trajectory in the gray matter of a long horizontal collateral (arrow) of the main axon, and the terminal field (asterisk) in layer III in the precentral gyrus. Small triangles represent large pyramidal cells in layer Vb. (B) Camera lucida two-dimensional reconstruction of terminal arbors in layer III provided by a long horizontal collateral of the main axon from a large, layer Vb pyramidal cell, shown in (A). In this reconstruction, terminal boutons and axon varicosities shown are larger than actual size. The same conventions were used in Fig. 4A and Fig. 5B.

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Fig. 3. (Continued)

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following, we describe the trajectory and terminal arbor of the thick collateral of the main axon. After leaving the main axon, the thick axon collateral ascended obliquely, anteriorly through layer Vb for 1.1 mm into the boundary between layers III and V, without any branches (Fig. 3A). Gradually shifting laterally, it proceeded anteriorly for 2 mm along the boundary between layers III and V, without any branches. At that point, the thick axon collateral abruptly turned dorsally towards the pial surface and, after going up for a short distance in the lower part of layer IIIc, it split into two thin branches, probably losing the myelin sheath (Fig. 3B). One branch (red) ascended vertically into layer IIIc, forming terminal arbors. The other branch (black) split into numerous branches in layer IIIc of which two long vertical branches extended into the middle of layer IIIc. One vertical branch (black) ascended into the lower part of layer IIIa+ b, forming terminal arbors. The other vertical branch (green) gave rise to a horizontal side branch 350 mm long, ramifying and forming terminal arbors in the upper part of layer IIIc, and extended into layer IIIa+ b near the border of layer II, issuing short side branches along the way. As shown in Fig. 2A, the two thin branches, two long vertical branches, and their side branches had varicosities (boutons en passant, thin arrows), spine-like protrusions (double arrows), and terminal boutons (arrowheads). Fig. 2A also shows terminal boutons surrounding the soma of unstained cells (asterisks). Since this axon collateral travelled within the cortical gray matter for 3 mm, this was designated as a long horizontal axon collateral. Viewed from the cortical surface, the terminal field in layer III of the above-mentioned long horizontal axon collateral was estimated to be a rectangle of 200 mm anteroposteriorly and 400 mm mediolaterally. It was located at a site 4.4 mm anterior to the central sulcus and 15 mm lateral to the medial margin of the hemisphere in the free surface of the precentral gyrus (hatched rectangle with 3, Fig. 1A). This site is 3 mm anterior and 1 mm lateral to the large pyramidal cell of origin in layer Vb.

3.3. A pyramidal cell in layer IIIB whose main axon projects to layers III and V In case 9657R, a pyramidal cell in layer IIIa+ b was labeled in the putative forelimb motor cortex on the free surface of the precentral gyrus (Fig. 1B; Fig. 2E; Fig. 4B). It had a medium-sized cell body (small and large diameters, 15×20 mm), and its apical dendrite divided into two shafts in layer III; only one shaft extended into layer I. The basal dendrites originated from the soma, but were incompletely labeled. The axon arose from the base of the soma and was covered by a myelin sheath 11 mm distant from the soma. It

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descended through layers III, V and VI towards the white matter, giving off many thin collaterals from the Ranvier nodes in layers III and V, but no collateral in layer VI. The axon entered the white matter at a point 250 mm medial to the soma of origin. It coursed downwards in the white matter for 1066 mm and, abruptly curving posteromedially, coursed for 1200 mm (Fig. 4B). The axon reached the anterior bank of the central sulcus. This trajectory of the axon is similar to U-fibers in the cerebral cortex. In the anterior bank of the central sulcus, the abovedescribed axon ascended vertically through layers VI and Vb towards the pial surface, shifting gradually medially and giving off tiny side branches (red, Fig. 4A). The axon became thin in the lower part of layer Vb, and, after ascending a little, divided into two branches. Each branch bifurcated in the middle part of layer V before entering layer Va. One branch terminated in layer V (black). The other three branches continued to ascend either obliquely or vertically in layers IIIc and IIIa+ b, issuing many shorter as well as longer side branches with terminal arbors (Fig. 4A). The two branches (black and blue) extended near layer II. Fig. 2B shows varicosities (arrows), spine-like protrusions, and terminal boutons (arrowhead) of the terminal arbors in layer III of this axon. Viewed from the cortical surface, a terminal field formed by the above-mentioned axon was estimated to be a rectangle of 300 mm mediolaterally and 580 mm anteroposteriorly. This terminal field was located at a site 800 mm medial and 6.8 mm along the cortical surface posterior to the pyramidal cell of origin in layer IIIB of M1 (hatched rectangle with 4, Fig. 1B)

3.4. An ascending axon branch arising from a pyramidal cell in layer IIIc that forms a terminal field in layer VI In case 9657R, a pyramidal cell in layer IIIc of the putative forelimb motor cortex was labeled within the halo of the injection site (Fig. 2F; Fig. 5A). The cell body was large (short and large diameters, 22× 32 mm), and a thick apical dendrite ascended to the border of layer II. An apical dendrite of a pyramidal cell that does not reach layers II or I was present in a pyramidal cell in layer III of the motor cortex of the cat (Keller et al., 1990). Basal and lateral dendrites were well developed and some dendrites extended into the core of the injection site. The main axon of this pyramidal cell emerged downward from a large lateral dendrite, and was covered by a myelin sheath about 13 mm distant from the soma. The main axon descended posterolaterally through layers IIIc, V and VI towards the white matter, without any collateral. It entered the white matter at a point 300 mm lateral and 1300 mm posterior to the soma of origin.

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As shown in Fig. 5A, the main axon travelled posteriorly through the white matter along the anterior bank, fundus, and posterior bank of the central sulcus. It could not be traced beyond the white matter under area 3b because of incomplete labelling. On coursing in the white matter immediately under the anterior bank of the central sulcus, the above-mentioned main axon issued one thick branch at a point 6 mm posterior to the soma of origin, measured along the cortical surface of the precentral gyrus (red stars, Fig. 5). This thick axon branch ran in the white matter

posteromedially for 200 mm to enter layer VI of the anterior bank of the central sulcus at the level of the lower one-third. Immediately after entering layer VI, the thick axon branch (arrow, Fig. 5B), referred to as the parent axon, turned horizontally in layer VI. The parent axon travelled within layer VI towards the fundus of the central sulcus for 1460 mm, gradually shifting medially, and then became a fine axon that ran for 170 mm and terminated near the border of area 3a. On its way to termination, the parent axon gave rise to a short, thick daughter branch (double arrow, Fig. 5B) at

Fig. 4. Trajectory and terminal arbors of the main axon from a pyramidal cell in layer IIIa +b of case 9657R. (A) Camera lucida two-dimensional reconstruction of terminal arbors in layers III and V provided by the main axon arising from a layer IIIa + b pyramidal cell, shown in B and Fig. 2E. (B) Brain diagram shows the position of the pyramidal cell of origin (blue triangle), the trajectory of its main axon (U-pathway), and the position of its terminal field in layers III and V in the anterior bank of the central sulcus.

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Fig. 5. Trajectory and terminal arbors of an axon branch of the main axon arising from a pyramidal cell in layer IIIc and coursing to the parietal cortex of case 9657R. (A) Brain diagram shows the position and size of the injection site, the position of the pyramidal cell of origin (blue triangle), and the trajectories of the main axon towards the parietal cortex and of its branch in the anterior bank of the central sulcus. The pyramidal cell of origin is shown in a photomicrograph of Fig. 2F. (B) Camera lucida two-dimensional reconstruction of terminal arbors of an axon branch of the main axon, shown in (A). The terminal field was restricted in layer VI in the anterior bank of the central sulcus. Red stars point to bifurcation of the main axon in the white matter.

1000 mm from its entry point into the anterior bank of the central sulcus. The parent axon created the first fine branch (black, Fig. 5B) ventrally at 246 mm from its entry point into the anterior bank of the central sulcus. This fine branch extended horizontally along the parent axon for 77 mm, giving rise to several short side branches with terminal arbors. Terminal boutons hanging like grapes from these side branches were also seen to extend over soma of unknown cells, possibly the terminal boutons made synaptic contacts on the soma (red asterisks, Fig. 5B). The parent axon created a second shorter, fine branch 323 mm from the first fine branch. After 140 mm from the second fine branch, the parent axon created the longest fine branch (green, Fig. 5B) running towards the white matter as well as a shorter fine branch towards layer V. The longest fine branch extended for 576 mm horizontally in layer VI, issuing ten smaller side branches at intervals of 23 – 92 mm. Each of these smaller side branches had terminal arbors with a diameter of approximately 30 mm. Fig. 2C and D illustrate the terminal boutons, varicosities, spine-like protrusions of one of these smaller side branches. In the remaining trajectory after the longest fine branch, the parent axon issued 19 fine branches in layer VI for a distance of 946 mm (2 longer and 17 shorter ones). The two longer fine branches (blue and red, Fig. 5B) also

ramified mainly along the parent axon, issuing a number of smaller side branches with arbors of diameters of 23–30 mm. The 17 shorter fine branches also formed terminal arbors of similar sizes. The daughter branch (double arrow, Fig. 5B) ascended anteriorly and obliquely in layer VI for 423 mm, and created six shorter fine branches. These six shorter fine branches formed terminal arbors around the daughter branch. Viewed from the cortical surface, the thick branch of the main axon of the above-mentioned layer IIIc pyramidal cell contained a terminal field consisting of arbors of the side branches in layer VI like a rectangle of 1,384 mm anteroposteriorly and 300 mm mediolaterally. This large terminal field was located in the anterior bank of the central sulcus at a site 600 mm medial and 8.4 mm along the cortical surface posterior to the pyramidal cell of origin in layer IIIc (hatched rectangle with 5, Fig. 1B).

4. Discussion The present study has revealed that some axons as well as long axon collaterals stemming from M1 of the macaque monkey course without axon branches towards their destinations in the same M1 horizontally

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through the gray matter or subcortically through the white matter. These local axons and long axon collateral form single terminal fields at the destination area of M1. Our findings indicate that there is a high degree of variability of projection pattern of local axons in local circuits within M1 in the monkey brain.

4.1. Pathways for long-range intrinsic connections within M1 The findings of the present study indicate that there are two pathways for long-range intracortical connections within M1; one is via the gray matter and the other via the white matter. The pathway via the gray matter is a horizontal connection. The main axons of pyramidal cells in layer III of the motor and somatosensory cortices of the monkey gave off long-range collaterals running horizontally in layers III or V (DeFelipe et al., 1986). In the prefrontal and visual cortices of both the monkey and humans, horizontally oriented axon collaterals from pyramidal cells travelled for a relatively long distance through the gray matter, and some of them formed periodic terminal patches (Burkhalter et al., 1993; Levitt et al., 1993; Kritzer and Goldman-Rakic, 1995; Fitzpatrick, 1996; Pucak et al., 1996; Melchitzky et al., 1998). In area 17 of the monkey several collaterals from a main axon of a pyramidal cell in layer III extended for 2 mm horizontally in layer III, forming four clusters of axon branches that ascended in layers III and II (McGuire et al., 1991). These longrange horizontal axon collaterals issued several side branches with clusters of terminal boutons on the way to their destinations; a fact referred to in others’ previous studies. In the present study, however, an axon collateral from a large pyramidal cell in layer Vb of the putative forelimb motor cortex travelled horizontally and anteriorly through the gray matter along the boundary between layers III and V for 3 mm, but it did not issue any side branches before forming a terminal field in layer III at the target area of M1 (Fig. 3). As far as we know, there is no other study of the long-range horizontal axon collateral like this in M1 in the monkey. The pathway via the white matter is one of the so-called U-fibres that are known to run just beneath the gray matter in the cerebral cortex and unite adjacent parts of the cerebral cortex. Recent studies with various tracers revealed axon trajectories similar to U-fibres in the prefrontal cortex (Levitt et al., 1993; Pucak et al., 1996; Melchitzky et al., 1998), in the somatosensory cortex (DeFelipe et al., 1986), in the motor cortex (Landry et al., 1980; Huntley and Jones, 1991), and in the auditory cortex (Ojima et al., 1992; Clarke et al., 1993). In the present study, two main axons from two pyramidal cells in layer III of the putative forelimb motor area on the free surface of the

precentral gyrus descended into the white matter. One main axon further descended into the white matter, curved abruptly medially, and entered the anterior bank of the central sulcus to form a terminal field in layers III and V (Fig. 4). The other main axon emitted a branch in the white matter, and this branch reached the anterior bank of the central sulcus to form a large terminal field in layer VI (Fig. 5). Thus, passing through the white matter, the two main axons unite one site in the putative forelimb motor area on the free surface and another site in M1 on the anterior bank of the central sulcus on the ipsilateral side.

4.2. Restriction of intracortical projections to specific layers of single cortical sites In the present study, two axons and one axon collateral arising from the putative forelimb area of M1 projected to particular layers of single sites in the same M1. One axon and one axon collateral formed single, small terminal fields in layers III and V and layer III in M1, respectively, and another axon branch formed a single, large terminal field in layer VI in M1. Axons of pyramidal cells with a single or two small terminal fields restricted to particular layers were observed in the visual cortex of the monkey and in the primary auditory cortex of the cat. In area 17 of the monkey brain, various types of neurons have axons or axon collaterals that project selectively to specific lamina of the same region (Fitzpatrick, 1996; Usrey and Fitzpatrick, 1996; Wiser and Callaway, 1996; Castellani and Bolz, 1997; Lund and Wu, 1997; Callaway, 1998). A similar pattern of axon projection was observed with associational connection between area 17 and 18 of the monkey. In the monkey, 18% of axons projecting from area 17 to 18 have one or two terminal ramifications in layer III, another 18% have a single terminal ramification in layer IV, and the majority (58%) have terminal ramifications in the upper half of layer IV and lower part of layer III in area 18. (Rockland and Virga, 1990). In the primary auditory cortex of the cat, 14% of intrinsic axons form a single terminal arbor in layer III in the adjacent part of the same region (Clarke et al., 1993). Thus, the laminar distribution of terminal fields of some types of axons projecting from the putative forelimb motor cortex to the same M1 seems to be governed by the same principles as those in the visual cortex of the monkey and in the auditory cortex of the cat. We observed that the thick branch of the main axon had a single large terminal field consisting of a number of small-sized axon arbors confined to layer VI (Fig. 5B). This axon branch is similar to the periodically branching axons in the primary auditory area of the cat (Ojima et al., 1991; Wallace et al., 1991; Ojima et al., 1992; Clarke et al., 1993), in area 17 of the cat (Gilbert

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and Wiesel, 1979; Martin, 1984; Kisva´rdy et al., 1986), and in the peristriate cortex of the monkey (Rockland, 1989; Rockland and Virga, 1989, 1990). These periodically branching axons provided side branches at regular intervals that also provided terminal arbors extending over two or three cortical layers. Laminar distribution of axon arbors, however, of the thick axon branch observed in the present study is different from those of earlier studies. It ran horizontally in layer VI for 1400 mm, giving off side branches at intervals of 100 mm, and each of the side branches provided terminal arbors spanning only layer VI. Similar axons with terminal fields restricted to a single cortical layer in the lateral agranular cortex corresponding to M1 of the rat have been studied by Aumann et al. (1998), using BDA. According to their study, two axons arising from the ventral lateral nucleus of the thalamus reach M1 via the internal capsule on the ipsilateral side, and each of them forms a single terminal field in layer V in M1. In other earlier studies on M1 of the monkey and cat following a single injection of HRP into a given representation site of M1, HRP-labeled dells and terminals were found in many sites within the major representation region involving the injection site (Huntley and Jones, 1991; Keller, 1993). This may imply that one representation site in M1 has reciprocal connections with various representation sites in the same region of M1, but it may not necessarily follow that a single neuron in the site injected with HRP projects to multiple sites in M1. The present study shows that three local axons arising from the putative forelimb area of M1 travelled for long distances without emitting any branches and that they formed single terminal fields within M1 on the same side. Similar axons with single terminal fields were also observed in area V2 in macaque monkeys; some axons arising from area V1 formed one or two terminal arbors in area V2 (Rockland and Virga, 1990). In conclusion, some pyramidal cells of M1 in the monkey project to single targets within the same M1 to form single terminal arbors.

Acknowledgements We wish to thank Dr M. Imada, S. Sawada, and R. Takagi for their assistance. This work was supported by the Investigate Program for Visiting Researchers of the Electrotechnical Laboratory, Grants-in Aid from the Ministry of Education, Science, Sports and Culture, Japan, and the Primate Research Institute, Kyoto University. References Arikuni, T., Yamashita, A., 1997. Local circuits of the motor cortex in adult macaque monkeys. Soc. Neurosci. Abstr., Vol.23, p.1273.

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