Terminal Organization of the Corticospinal Projection from the Arm/hand Region of the Rostral Primary Motor Cortex (M1r or Old M1) to the Cervical Enlargement (C5-T1) in Rhesus Monkey

Abstract

High resolution anterograde tracers and stereology were used to study the terminal organization of the corticospinal projection (CSP) from the rostral portion of the primary motor cortex (M1r) to spinal levels C5-T1. Most of this projection (90%) terminated contralaterally within laminae V-IX, with the densest distribution in lamina VII. Moderate bouton numbers occurred in laminae VI, VIII and IX with few in lamina V. Within lamina VII, labeling occurred over the distal-related dorsolateral subsectors and proximal-related ventromedial subsectors. Within motoneuron lamina IX, most terminations occurred in the proximal-related dorsomedial quadrant, followed by the distal-related dorsolateral quadrant. Segmentally, the contralateral lamina VII CSP gradually declined from C5-T1 but was consistently distributed at C5-C7 in lamina IX. The ipsilateral CSP ended in axial-related lamina VIII and adjacent ventromedial region of lamina VII. These findings demonstrate the M1r CSP influences distal and proximal/axial related spinal targets. Thus, the M1r CSP represents a transitional CSP, positioned between the caudal M1 (M1c) CSP which is 98% contralateral and optimally organized to mediate distal upper extremity movements (Morecraft et al., 2013), and dorsolateral premotor (LPMCd) CSP being 79% contralateral and optimally organized to mediate proximal/axial movements (Morecraft et al., 2019). This distal to proximal CSP gradient corresponds to the clinical deficits accompanying caudal to rostral motor cortex injury. The lamina IX CSP is considered in the light of anatomical and neurophysiological evidence which suggests M1c gives rise to the major proportion of the cortico-motoneuronal (CM) projection, while there is a limited M1r CM projection.

1.0. INTRODUCTION

Much of the corticospinal projection (CSP) arises from extensive regions of the frontal lobe and plays an important role in the generation and control of movement. Although the frontal CSP subserves many motor functions, the part forming the caudal region of the primary motor cortex (M1c) - residing within the anterior bank of the central sulcus - has attracted considerable attention because in some primates, it harbors cortico-motoneuronal (CM) projection neurons (Rathelot & Strick, 2006, 2009), which are implicated in skilled hand use. As the name implies, this projection originates from cortical neurons whose descending axons make direct synaptic contacts with motoneurons residing in spinal lamina IX, and possibly their dendrites extending beyond lamina IX. The CM projection is considered to be a relatively recent evolutionary feature of the primate motor system, which allows motor cortex direct influence over brainstem and spinal motoneurons, bypassing local, segmental reflex mechanisms. In macaques, CM cells in the hand/arm region of M1c exert direct influence over intrinsic hand and other forearm muscles, and are particularly active during tasks such as precision grip, characterized by a “fractionated” pattern of muscular activity necessary for independent digit movements (Kirsch et al., 2014; Lemon, 1988; Muir & Lemon, 1983; Quallo et al., 2012; Rathelot & Strick, 2006; Schieber, 1995, 2011).

In the macaque, the CSP from M1c has also been extensively studied using anterograde neuroanatomical approaches to not only examine the topography of the CM projection to lamina IX, but to study the entirety of the M1c CSP to other spinal cord laminae as well (Coulter & Jones, 1977; Hoff, 1934; Kuypers & Brinkman, 1970; Liu & Chambers, 1964; Maier et al., 2002). A study from our laboratory quantified the terminal organization of the CSP from the arm/hand area of M1c to spinal levels innervating upper extremity muscles (C5-T1) (Morecraft et al., 2013). Several structural characteristics were found, pointing to the conclusion that the M1c CSP was optimally organized to mediate distal movements of the contralateral upper extremity including grasping and manipulation. A principal finding was that a remarkable 98% of the total labeled boutons counted were observed to terminate in the contralateral cord, with only 2% of boutons located ipsilaterally. The projection to lamina VII of the intermediate zone was most prominent, constituting 59% of the contralateral CSP, targeting the dorsolateral sectors of lamina VII. However, the second highest proportion of boutons was found amongst the motor nuclei of lamina IX (18%), suggesting the CM projection is a significant component of the total M1c CSP. Further analysis suggested that the M1c CM projection may influence motoneurons innervating the flexors acting on the shoulder and elbow located rostrally (C5-C7) along with the flexors, extensors, abductors and adductors acting on the digits, hand and wrist located more caudally (C8-T1) (Jenny & Inukai, 1983).

In a more recent investigation , we quantified the terminal organization of the CSP from the rostrally located lateral premotor cortex (LPMC), which contrasts heavily with the M1c CSP (Morecraft et al., 2019). Most of the projections from the ventral part of the lateral premotor cortex (LPMCv) did not extend below C2. However, a relatively prominent CSP to C5-T1 originated from the arm-shoulder area of the dorsolateral premotor cortex (LPMCd). This CSP was found to be bilateral, with 79% boutons found contralateral. The projection to lamina VII constituted 63% of the total contralateral projection, heavily targeting the ventromedial sectors. Notably, these sectors contain propriospinal neurons with long-range bilateral axon projections (Matsushita et al., 1979; Molenaar & Kuypers, 1978). Surprisingly, the second highest proportion of contralateral boutons was found in lamina VIII (23%), which harbor this same propriospinal neuron type, but also axial motoneurons (Sprague, 1948). Compared with M1c, contralateral lamina IX labeling was light (3%) targeting the dorsomedial quadrant, where proximal upper limb flexor motoneurons reside (Jenny & Inukai, 1983). Segmentally, the CSP to contralateral laminae VII and IX preferentially innervated C5-C7, which supplies shoulder, elbow, and wrist musculature. These findings clearly demonstrated that the LPMCd CSP influences axial and proximal upper limb movements.

What remains to be investigated is the terminal organization of CSP from lateral frontal cortex residing between M1c and LPMCd. This cortex is identified as the rostral part of M1 (M1r) or “old” M1 (Rathelot & Strick, 2009). Anatomically, it is possible that the M1r CSP takes the form of a transitional terminal pattern, with some characteristics reflecting the distal-related M1c CSP, and others the proximal/axial-related LPMCd CSP. Regarding the contralateral lamina VII projection, a transitional pattern might include dense terminals in the dorsolateral region (as with M1c), as well as the ventromedial region (as with LPMCd). From the perspective of the CM projection to lamina IX, retrograde transneuronal tracer studies in macaque monkey suggest that CM neurons are abundant in M1c but are scarce in M1r (Rathelot & Strick, 2006, 2009). In agreement with the M1c findings, a recent electrophysiological study reported that focal M1c stimulation produced monosynaptic responses in contralateral forelimb motoneurons with a range of latencies, with the fastest mediated by rapidly conducting CM neurons (Witham et al., 2016). However, stimulation of M1r also gave rise to monosynaptic responses, albeit considerably slower and smaller than those from M1c, and possibly mediated by more slowly conducting CM neurons . This important finding could be supported by the demonstration of M1r CSP terminations within motoneuron lamina IX. Therefore, the present study was undertaken to determine the terminal organization of the M1r CSP to spinal levels C5-T1 using high-resolution anterograde tract tracers and stereological methodology in the rhesus macaque monkey.

2.0. MATERIALS AND METHODS

The terminal distribution of the corticospinal projection (CSP) arising from the rostral part of the primary motor cortex (M1r) to spinal levels C5-T1 was studied in 3 rhesus monkeys (Macaca mulatta) (Fig. 1; Table 1) using high resolution dextran tract tracers and stereology. Case SDM57-LYD listed in Table 1 was also used in our previous study on the M1c CSP (Morecraft et al., 2013) but only to verify general M1 CSP topography. Importantly, it was not used in the quantitative (stereological) analysis of the M1c CSP because the main body (and core) of the injection site was located on the gyral surface, thus corresponding to M1r (see Results). All experimental protocols were approved by The University of South Dakota Institutional Animal Care and Use Committee. All experimental and surgical procedures were conducted at the University of South Dakota and followed United States Department of Agriculture, National Institutes of Health, and Society for Neuroscience guidelines for the ethical treatment of animals.

Figure 1.

Line drawings of the lateral surface of the cerebral cortex in cases SDM57-LYD, SDM89-FD, and SDM92-FD depicting the respective lucifer yellow dextran (LYD, yellow) and fluoresceine dextran (FD, green) injection sites in the cortical arm/hand region of M1r. On the full lateral views, the irregular shaped black line within the LYD and FD injection sites represents the boundary between the centrally located injection site core, and peripherally located injection site halo. The three small white dots on each injection site core represent the Hamilton needle penetration locations through which each respective tract tracer was delivered. The pullout depicts the physiological map of movement representation obtained with intracortical microstimulation used to localize the arm/hand representation of M1r prior to injection of each respective tract tracer. Abbreviations: cs, central sulcus; Di, digit; ec, ectocalcarine sulcus; El, elbow; F, face; Hp, hip; ilas, inferior limb of arcuate sulcus; ios, inferior occipital sulcus; ips, intraparietal sulcus; J, jaw; K, knee; L, leg; ls, lunate sulcus; lf, lateral fissure; LL, lower lip; ps, principle sulcus; Sh, shoulder; slas, superior limb of the arcuate sulcus; sts, superior temporal sulcus; Th, thumb; UL, upper lip; Wr, wrist.

Table 1.

Description of the Experimental Parameters in each Case

Case	Sex	Age (yrs.)	Weight (kg)	Area Injected	Tracer/Injections	Total Volume (μL)	Injection Core Vol. (mm3)	Injection Halo Vol. (mm3)	Post-injection survival (days)
SDM57LYD	female	18.0	6.0	M1r arm	LYD/3	1.2	8.34	70.2	32
SDM89FD	female	1.0	2.3	M1r arm	FD/3	1.2	7.5	40.6	33
SDM92FD	male	1.2	3.3	M1r arm	FD/3	1.2	3.2	32.4	33

2.1. Neurosurgery, Intracortical Microsimulation and Tract Tracer Injection

Preoperatively, each monkey was immobilized and sedated ketamine hydrochloride (10mg/kg) and administered atropine (0.5mg/kg). Following intubation, each subject was placed on a mechanical ventilator and anesthetized with a mixture of 1.0–1.5% isoflurane and surgical grade air/oxygen. Once deeply anesthetized, each animal was placed in a neurosurgical head holder and mannitol was administered intravenously (1.0–1.5g/kg) to reduce overall cortical volume and enhance surgical accessibility of the brain. Under sterile conditions and isoflurane anesthesia, a skin flap was made over the cranium followed by an oval bone flap over the precentral cortical region. In all cases the precentral region, extending from the central sulcus to the cortex forming the arcuate sulcus, was mapped using intracortical microstimulation (ICMS) as previously described under ketamine anesthesia (McNeal et al., 2010; Morecraft et al., 2013) (Fig. 1). Current intensity ranged between 7 and 90 μA. Threshold currents were determined, and evoked movements were discrete isolated twitches, confined to specific body parts. The specific movements were recorded when agreed upon by at least two observers. After ICMS mapping, isoflurane anesthesia was resumed and the anterograde neural tract tracer lucifer yellow dextran (LYD) or fluorescein dextran (FD) (Molecular Probes, Eugene, OR) was injected into sites within M1r that were surrounded by well-defined upper extremity motor responses. The tracer biotinylated dextran amine (BDA) was injected into another cortical area of interest. Graded pressure injections with a Hamilton microsyringe were made approximately 1.5–2.5 mm below the pial surface with a total volume of 1.2 μL (Table 1). The Hamilton microsyringe was held in a specially designed microdrive attached to a stabilized electrode micromanipulator (model 1460 Kopf Instruments, Tujunga, CA) and all injection penetrations were made perpendicular to the cortical surface. Three injections were placed in a triangular pattern approximately 1.5 mm apart. Following the injection procedure, the surgical field was irrigated with 0.9% sterile saline then gently swabbed. The dura was repositioned, closed with sutures and the bone flap replaced and anchored. The temporalis muscle was sutured in place and the skin was closed using standard surgical technique. The animal was carefully monitored after surgery and penicillin (procaine G) was used as pre- (24 hours prior to surgery) and post- (for 9 days after the surgery) operative prophylaxis antibiotic. Buprenorphine (0.01 mg/kg) was used as a post- (48–72 hours after surgery) operative analgesic.

2.2. Tissue Processing

Following a survival period of 32–33 days after tract tracer injection, each monkey was anesthetized with an IP overdose of pentobarbital (50 mg/kg or more) and perfused transcardially with 0.9% saline. Saline infusion was followed by 2 liters of 4% paraformaldehyde in 0.1M phosphate buffer at pH 7.4 (PB) to fix the tissue. The first liter of paraformaldehyde fixative was administered rapidly and the second liter via slow drip. Then infusion of one liter each of 10% sucrose in PB followed by 30% sucrose in PB for tissue cryoprotection concluded the perfusion. The entire central nervous system was removed, placed in 30% sucrose in PB and stored for 2 to 5 days at 4° C. During removal of the spinal cord region, several millimeters of each dorsal and ventral spinal root were intentionally left on the cord. Ventral and dorsal roots of the spinal cord were extended so their positions relative to the long axis of the cord could be clearly recorded for determining the segmental level locations and intersegment boundaries during the data analysis and reconstruction process. Prior to, and after blocking the tissue into segments for microtome sectioning, the cortex, brainstem and spinal cord were photographed with metric calibration.

In all cases, the cerebral cortex was frozen sectioned in the coronal plane on a sliding microtome (American Optical 860, Buffalo, NY, USA) at a thickness of 50 μm in cycles of 10, forming 10 complete series of evenly spaced tissue sections respectively. Each spinal cord was blocked from the lower medulla, perpendicular to its long axis, frozen with dry ice and cut transversely (90° to its long axis) on the sliding microtome at a thickness of 50 μm in cycles of 6 or 8, with each forming a complete series of evenly spaced tissue sections respectively. For both the cortical and spinal cord sections, one series of tissue sections was mounted on subbed slides, dried and eventually stained for Nissl substance using thionin and coverslipped with Permount to evaluate cytoarchitectonic organization (Morecraft et al., 2013; Morecraft et al., 2012).

A single series of tissue sections through the cortex and spinal cord were then processed using single labeling immunohistochemistry to visualize BDA (i.e., BDA alone) (Morecraft, McNeal, Stilwell-Morecraft, Dvanajscak, et al., 2007) (Figs. 2, 3). To accomplish this, one complete series of tissue sections from the cortex and spinal cord was used to process BDA using the Vectastain Elite avidin-biotin complex (ABC) labeling procedure (PK-6100, Vector Laboratories, Burlingame, CA, USA) (RRID:AB_2336819). Briefly, the tissue sections were rinsed in 0.05M tris buffered saline at pH 7.4 (TBS) then incubated overnight at 4° C in TBS with 5% normal goat serum (NGS) and 1.25% Triton X-100. Next, the sections were rinsed in TBS and then incubated in the ABC solution for 4 hours at room temperature. The sections were then rinsed with TBS and incubated in a 0.05% solution of 3, 3’ diaminobenzidine tetrahydrochloride (DAB) (08980681, MP Biochemicals, Ohio) for approximately 10 minutes. Subsequently, 30% hydrogen peroxide (H₂O₂) was added to the DAB solution achieving a final H₂O₂ concentration of 0.012%. The tissue was incubated in the DAB/H₂O₂ solution for another 8–10 minutes yielding an insoluble brown reaction product and immediately placed in TBS to stop the reaction. Following this processing, the BDA-stained tissue sections were then rinsed in TBS, mounted on subbed slides, dried, then dehydrated in graded alcohol solutions, cleared in xylene and coverslipped using Permount.

Figure 2.

Plate of low-power digital photomicrographs of immunohistochemically processed coronal tissue sections through the cerebral cortex illustrating the respective lucifer yellow dextran (LYD) and fluoresceine dextran (FD) injection sites in cases SDM57-LYD (a), SDM89-FD (b), and SDM92-FD (c). On each injection site, the dashed line represents the external boundary of the estimated injection site core and the dotted line the external boundary of the estimated injection site halo which were both determined using microscopic analysis. The scale bar in the bottom left of panel (a) = 2 mm and applies to all panels. The anatomical orientation compass in panel (c) also applies to panel (b). Abbreviations: cs, central sulcus; d, dorsal; l, lateral; m, medial; spcd, superior pre-central dimple; v, ventral.

Figure 3.

Plate of high-power digital photomicrographs of immunohistochemically processed tissue sections through the spinal cord illustrating dextran labeled terminal axon fibers and boutons (black arrowheads) in the contralateral gray matter of cases SDM57-LYD (a and b), SDM89-FD (c and d), and SDM92-FD (e and f). The white arrowheads in panels c, e and f, show examples of terminal boutons over the soma or proximal dendrites of lamina IX motoneurons. In all panels, the blue colored fibers and terminals are from the respective M1r injection sites. Brown colored fibers and terminals are from a biotinylated dextran amine injection site placed in another cortical region of interest. In the photographs with the insets, the asterisk denotes the location on the main panel when applicable. (a) Section through C5 showing terminal labeling in the central region of lamina VII. (b) Section though C6 showing terminal labeling in the ventrolateral region of lamina VII. (c) Section through C5 showing terminal labeling in the ventrolateral part of lamina VII and the adjacent dorsomedial region of lamina IX. Note, in the inset the blue bouton clusters can be seen over a motoneuron soma in lamina IX. (d) Section through C7 showing terminal labeling in the dorsolateral region of lamina VII. The inset shows dense terminal labeling in the medial region of lamina VII. (e) Section though C5 showing terminal labeling in the ventrolateral part of lamina VII and dorsomedial region of lamina IX. The 2 insets were taken at different focal planes to more clearly show the terminal boutons to the left of the larger motoneuron (left inset) and the terminal boutons to the right of the same motoneuron (right inset). (f) Section through C6 showing terminal labeling in the ventrolateral part of lamina VII and dorsal region of lamina IX. The inset shows terminal labeling in proximal/axial related lamina VIII at C8. The micron bar in the bottom right of panel f = 50 μm and applies to all main panels (all taken with at 40x). The insets were all captured at 60x. Roman numerals represent Rexed’s laminae.

Next, two additional and separate series of tissue sections was used for double labeling in which both BDA+LYD, or BDA + FD were visualized employing a simple multiple colorimetric detection method (Figs. 2, 3) (Morecraft, McNeal, Stilwell-Morecraft, Dvanajscak, et al., 2007; Morecraft, 2014). To accomplish this, BDA was reacted first in a full series of tissue sections according to the above ABC labeling protocol staining BDA brown with DAB. The same tissue sections were then rinsed in TBS and incubated in TBS with 5% NGS and 1.25% Triton X-100 overnight. The tissue sections were then transferred and incubated in 5% goat serum in TBS with biotinylated anti-lucifer yellow directed against LYD at a dilution of 1:200 (A5751, Molecular Probes, Eugene, OR) or biotinylated anti fluorescein directed against FD at a dilution of 1:500 (BA-0601, Vector Laboratories) for approximately 40 hours at 4° C. The tissue was then rinsed in TBS and incubated in a solution of ABC for 4 hours at room temperature, rinsed again in TBS and incubated with the Vector SG peroxidase substrate kit (SK-4700, Vector laboratories) for approximately 5–10 minutes yielding a blue reaction product for the second tracer (LYD or FD). The sections were rinsed, mounted on subbed glass slides, dried, dehydrated and coverslipped using Permount. Thus, BDA was stained brown and LYD or FD was stained blue in the same tissue sections (Fig. 3). Finally, in 2 additional animals that did not receive motor cortex injections, we studied NeuN stained spinal cord sections to assist in the analysis of identifying Rexed’s laminae (Morecraft et al., 2013, see Fig. 5). The NeuN immunohistochemical and tissue processing procedures that were followed have been provided in our previous reports (Morecraft et al., 2013; Morecraft et al., 2012).

Figure 5.

(a) Percentage of total estimated boutons distributed in the contralateral (blue bars) and ipsilateral (red bars) spinal laminae in segments C5 – T1. Each bar is the percentage of boutons from each individual monkey (SDM57, SDM89, SDM92) and the average of all three monkeys (far right) as indicated on the abscissa. Almost 90% of all labeled boutons were located in the contralateral spinal cord. Estimated number of contralateral (b) and ipsilateral (c) boutons within Rexed’s lamina of C5 – T1 in each individual monkey case and the average of three monkeys as indicated on the abscissa. Each bar represents the stereological estimate of the total number of boutons in the dorsal horn (lamina I-IV), intermediate zone (laminae V, VI, VII and VIII and RMB), ventral horn (lamina IX) and centrally located lamina X in C5 – T1. The number above each bar represents the percentage of all contralateral (b) or ipsilateral (c) boutons located in each lamina (I-X) or region (RMB) for each monkey case and for the average obtained from all three monkeys. Note the different ordinate scales in (b) and (c). Error bars on the average data are 1 SEM.

2.3. Data Analysis

Localization of the cortical injection site and of the terminal boutons within the spinal gray matter (C5-T1) was accomplished using brightfield illumination on a BX-51 Olympus microscope (Leeds Precision Instruments, Minneapolis, MN). Attached to the microscope was a high resolution MAC 5000 motorized stage (Ludl Electronic Products, Hawthorne, NY, USA) which was joined to the Neurolucida (RRID:SCR_001775) and Stereo Investigator (RRID:_SCR002526) neuroanatomical data collection software (Microbrightfield, Inc., (RRID:_SCR_004314), Colchester, VT, USA) in a Dell Precision Tower 5810. The Neurolucida system was used to plot the major anatomical structures and their boundaries in Nissl and immunohistochemical stained tissue sections and record the locations of the cortical injection site and distribution of terminal-like profiles (boutons) in the spinal cord in immunohistochemically processed tissue sections (Fig. 4). The cortical injection sites in M1r were localized by plotting the external boundary of the core region and external boundary of the halo region and determined as previously described (Morecraft et al., 2019) (Figs. 1, 2).

Figure 4.

Line drawings depicting selected horizontal sections showing the topography of terminal labeling within Rexed’s laminae through spinal levels C5 to T1 in cases SDM57-LYD (left), SDM89-FD (center) and SDM92-FD (right). The locations of labeled axons in the lateral corticospinal tract and other white matter regions are identified by the green dots. The locations of terminal boutons and bouton clusters within Rexed’s laminae are identified by the blue dots. Roman numerals in section C5 of SDM57-LYD designate Rexed’s laminae and their respective subdivisions and apply to all spinal sections. Note the contralateral CSP in cases SDM89 and SDM92 was actually on the right side of the spinal cord, since the cortical FD injection was placed in the left hemisphere (see Fig. 1). However, for comparative purposes with case SDM57, the spinal cord sections for cases SDM89 and SDM92 have been flipped in this diagram.

Using immunohistochemically processed spinal cord sections, terminal boutons were plotted in every other tissue section to obtain a general characterization of the topography and relative density of the projection in Rexed’s laminae with the aid of Olympus UPlan Apo 20x–40x microscope objectives (Leeds Precision Instruments, Minneapolis, MN) (Fig. 4). Matching Nissl stained sections were used to define laminar gray matter compartmentalization within each tissue section as well as determine the segmental level boundaries within the longitudinal series of tissue sections. As detailed in our previous report, most of Rexed’s laminae on both sides of the spinal cord were subdivided into Regions of Interest (ROI) (Morecraft et al., 2013). Specifically, laminae I-VI were divided into medial and lateral subsectors, lamina VII into dorsolateral, dorsomedial, ventrolateral, ventromedial and ventral subsectors and lamina IX was divided into quadrants. Lamina X was divided into a contralateral and ipsilateral half, lamina VIII was not subdivided, and a reticulated marginal border (RMB) was recognized on both sides of the spinal cord. The RMB is an area identified by Rexed (Rexed, 1954) and Kuypers (Kuypers, 1981) as possessing extensive dendritic arbors of neurons located in the lateral part of laminae V-VII which protrude into the dorsolateral funiculus. This area also corresponds to the location noted by Kuypers to contain “spinal border cells” (sbc) of the propriospinal system (see Figs 1, 3 in (Molenaar & Kuypers, 1978)).

The primary motor and lateral premotor regions and adjacent parietal cortex boundaries were evaluated for cytoarchitecture according to the criteria of Pandya and colleagues (Morecraft et al., 2004; Morecraft et al., 2012; Morecraft, Stilwell-Morecraft, et al., 2015; Pandya, 2015). Specifically, the cytoarchitectural organization (areas 4, 6DC, 6DR, 6Va, and 6Vb) was microscopically determined and superimposed on the reconstructed injection site location.

2.4. Stereological Data Analysis

Using Stereo Investigator 7 (MicroBrightField, Inc., (RRID:SCR_001775, RRID:_002526), Williston VT, USA), unbiased estimates of terminal boutons were obtained in the spinal gray matter ROI’s using the 3-D Optical Fractionator probe as previously described (McNeal et al., 2010; Morecraft et al., 2013; Morecraft, McNeal, Stilwell-Morecraft, Gedney, et al., 2007) and our estimation of the cortical injection site volume (halo and core) was achieved by applying the Cavalieri probe as previously reported (Morecraft et al., 2013; Pizzimenti et al., 2007). Stereology was applied in every other tissue section through each series of cortical (injection site volume estimation) and spinal cord (terminal bouton number estimation) tissue sections. For terminal bouton estimation, unbiased sampling was applied within in each ROI such that each location along the tissue section axis had an equal probability of being included in the sample and all locations in the plane of section (excluding the set guard zones) had an equal probability of being sampled with the probe (Gundersen, 1986; Napper, 2018; Sterio, 1984; West, 2012; West et al., 1991). Counting rules were applied across all case material so that all boutons had equal probabilities of being counted. The use of the applied 3-D probe avoids sampling biases, and the most important feature of these probes is that they are not affected by variations in size, shape, orientation and distribution of the biological structures/particles of interest (Olesen et al., 2017; West, 2012). Therefore, the fact that the CSP is randomly distributed throughout the laminae did not affect the estimation process of total bouton number. The design of our stereological application (e.g., bouton definition, choice of dissector probe for quantifying bouton numbers and injection site volumes, counting frame size, guard zone depth, etc.) and technical issues related to our use of multiple dextran tracers and experimental strategies have been discussed in detail in our previous papers (McNeal et al., 2010; Morecraft, Ge, et al., 2015; Morecraft et al., 2016; Morecraft et al., 2013; Morecraft, McNeal, Stilwell-Morecraft, Gedney, et al., 2007; Morecraft et al., 2021).

In all cases, microscopic identification/counting of all terminal boutons was accomplished using an Olympus PlanApo 100x oil objective (Leeds Precision Instruments, Minneapolis, MN). For counting terminal boutons, the main stereological parameters included the counting brick dimensions, tissue thickness, counting brick placement, guard zones and disector height. The same counting frame (109.2/71.4 μm) and X/Y grid placement (125.3/241.9 μm) was applied to all case material as in our previous CSP reports on M1, supplementary motor area (M2 or SMA) and LPMC ((McNeal et al., 2010; Morecraft et al., 2013; Morecraft et al., 2019). Tissue thickness was determined by sampling 5 random gray matter sites in every other section then computing an average from this data.

In the spinal cord we estimated bouton numbers in all Rexed’s lamina (Fig. 5) and respective sub compartments (Figs. 4, top left, 6, 7). In addition, we conducted a contralateral and ipsilateral segmental analysis for total bouton number at each segmental level (C5, C6, C7, C8 and T1) and performed a segmental analysis for terminal boutons in contralateral laminae VII and IX for all M1r cases (Fig. 7).

Figure 6.

(a) Estimated number of contralateral boutons within each subsector of lamina VII in C5 – T1 for each individual monkey (SDM57, SDM89, SDM92) and the average of all three (far right) as indicated on the abscissa. (b) Estimated number of contralateral boutons within each subsector of lamina IX in C5 – T1 in each individual monkey case and the average of all three as indicated on the abscissa. (c) Estimated number of ipsilateral boutons within each subsector of lamina VII in C5 – T1 for each individual monkey and the average of all three as indicated on the abscissa. (d) Estimated number of ipsilateral boutons within each subsector of lamina IX in C5 – T1 in each individual monkey and the average of all three as indicated on the abscissa. Note the different ordinate scales in (a) and (b) (contralateral projection) versus (c) and (d) (ipsilateral projection). In each panel, the number above each bar is the percentage of either all contralateral or ipsilateral boutons located lamina VII and IX for that individual case or for the computed average obtained from all three monkeys. Error bars on the average data sets in all panels are 1 SEM. Abbreviations: DM, dorsomedial; DL, dorsolateral; VM, ventromedial; VL, ventrolateral; V, ventral.

Figure 7.

Graphs showing the data obtained from our segmental analysis. Estimated number of contralateral (a) and ipsilateral (b) boutons in all laminae of each spinal segment (C5 – T1). Each bar is the stereological estimate of the number of boutons in all laminae of each spinal segment (C5 – T1) from each individual monkey (SDM57, SDM89, SDM92) and the average of all three monkeys (far right) as indicated on the abscissae. The number above each bar represents the percentage of boutons for that monkey or average percentage from all three monkeys. Estimated number of contralateral (c) and ipsilateral (d) boutons in the various subsectors of lamina VII of each spinal segment (C5 – T1). The black bars represent the average total number of boutons for each segmental level. Note the decline in total number of boutons progressing caudally. Estimated number of contralateral (e) and ipsilateral (f) boutons in the various subsectors of lamina IX of each spinal segment (C5 – T1). The black bars represent the average total number of boutons for each segmental level. Note the different ordinate scales in contralateral (a, c and d) and ipsilateral (b, d and f) projections. Error bars on the average data sets are 1 SEM. Abbreviations: DM, dorsomedial; DL, dorsolateral; VM, ventromedial; VL, ventrolateral; V, ventral.

2.5. Data Reconstruction and Presentation

Publication quality images of injection sites, labeled fibers and labeled boutons were captured using a Spotflex 64 Mp shifting pixel camera, (Diagnostic Instruments Inc., Sterling Heights, MI, USA, version 4.6), mounted on an Olympus BX51 microscope. Photographic montages of the injection sites and labeled fibers were created using Adobe PhotoShop 7.0 (Adobe Systems Inc., San Jose, CA, USA) (Figs., 2, 3). Brightness and contrast were adjusted in the images. Cortical reconstructions were developed as previously described using metrically calibrated digital images of the cortical surface (Morecraft & Van Hoesen, 1992). Publication quality illustrations were created using Adobe Illustrator and Adobe Photoshop (Adobe Systems Inc., San Jose, CA, USA) (e.g., Figs. 1–4).

3.0. RESULTS

Histological analysis of the 3 cortical injection sites revealed that all were located on the lateral surface of M1, corresponding to the gyral, or rostral part of M1 (M1r) and in no case did the injection site spread inferiorly to involve subcortical gray matter structures. In case SDM57-FD some of the peripheral region of the injection site halo involved area 4 in the anterior bank of the central sulcus. But the main portion of the injection site including the core, was located on the gyral region of area 4, corresponding to M1r (Figs. 1, 2a). The injection site halo and core in cases SDM89-FD and SDM92-FD were both localized to the gyral portion of area 4, corresponding to M1r (Figs 1, 2b, c). In all cases, the injection sites involved layer V which harbors the pyramidal cell bodies giving rise to corticospinal axon projections (Kuypers, 1981; Martin, 2005). In all cases, tracer from the injection sites was transported below the most inferior spinal level of interest (T1).

The estimated numbers of M1r labeled terminal boutons within each ROI (with the exception of the subsector breakdown for labeling in laminae VII and IX) for SDM57, SDM89 and SDM92 are presented in Table 2 (contralateral projection) and Table 3 (ipsilateral projection). Below we report the data representing the mean of all 3 M1r cases and the general distribution patterns of bouton labeling were similar across the three cases. Individual differences between the cases can be seen in Tables 2 and 3 and in Figures 5–6.

Table 2.

Contralateral Bouton Counts in Each Control Case by Spinal Lamina¹

Case	Total	I-III		IV		V		VI		RMB	VII	VIII	IX	Xc
		Med	Lat	Med	Lat	Med	Lat	Med	Lat
SDM 57	28,276	0	0	0	0	136	0	1,430	4,702	1,021	17,923	1,362	1,634	68
						(0.5)		(5)	(17)	(4)	(63)	(5)	(6)	(0.2)
SDM 89	42,911	0	0	0	0	0	296	653	2,674	2,971	24,433	3,328	7,963	593
						(0)	(.7)	(2)	(6)	(7)	(57)	(8)	(19)	(1)
SDM 92	60,902	0	0	0	0	0	346	1,270	3,351	1,731	43,232	2,714	5,717	2,541
							(0.6)	(2)	(6)	(3)	(71)	(4)	(9)	(4)
Mean	44030	0	0	0	0	45.3	214	1118	3576	1908	28529	2468	5105	1067
		(0.)	(0)	(0)	(0)	(0.1)	(0.5)	(3)	(8)	(4)	(65)	(6)	(12)	(2)

¹Percentages of total are reported in parentheses.

Values are rounded to the nearest whole number, except when the value is 0.7 or less. Lat, lateral; Med, medial; RMB, reticulated marginal border.

Table 3.

Ipsilateral Bouton Counts in Each Control Case by Spinal Lamina¹

Case	Total	I-III		IV		V		VI		RMB	VII	VIII	IX	Xi
		Med	Lat	Med	Lat	Med	Lat	Med	Lat
SDM 57	1,623	0	0	0	0	0	0	0	0	0	272	1,293	0	68
											(17)	(80)		(4)
SDM 89	4,145	0	0	0	0	0	0	0	0	0	1,423	2,258	237	236
											(34)	(54)	(6)	(6)
SDM 92	12,523	0	0	0	0	0	0	0	0	0	5,192	5,547	1,209	575
											(41)	(44)	(10)	(5)
Mean	6100	0	0	0	0	0	0	0	0	0	2298	3033	482	293
											(38)	(50)	(8)	(5)

¹Percentages of total are reported in parentheses.

Values are rounded to the nearest whole number, except when the value is 0.7 or less. Lat, lateral; Med, medial; RMB, reticulated marginal border.

In terms of laterality, the heaviest amount of terminal bouton labeling was found in the contralateral spinal gray (90%) with fewer labeled terminals occurring ipsilaterally (10%) (Fig. 5a). Below we present our findings in order of highest to lowest labeling density for each major spinal region (Fig. 5a, c). This includes a breakdown of labeling within the various subsectors of laminae VII and IX (Fig. 6) and a segmental analysis (Fig. 7)

3.1. Contralateral M1r Projection

3.1.1. Overall Laminar Organization of Terminal Labeling

With respect to the entire contralateral projection, 87% of the total number of contralateral terminal boutons was found in the intermediate zone (laminae V, VI, VII, VIII and the RMB) (Fig. 5b; Table 2). Fewer terminations (12%) were found in the contralateral ventral horn (lamina IX), and no terminals were detected in the contralateral dorsal horn (lamina I-IV). A sparse terminal projection (2%) was distributed in the contralateral half of midline lamina X.

3.1.2. Intermediate Zone Labeling

Within the intermediate zone, the highest percentage of labeled terminal boutons, and hence strongest CSP to the intermediate zone, was located in lamina VII (65%) (Figs. 3, 5b). Within lamina VII, consistent labeling was distributed over both lateral and medial subsectors and this diffuse pattern extended to involve the ventral subsector (Fig. 6a). For example, the ventrolateral and dorsolateral subsectors on average contained 28% and 22% of estimated boutons respectively. Similarly, the ventromedial and dorsomedial subsectors contained 20% and 16% of the detected boutons respectively, with 14% distributed over the ventral subsector.

A much smaller and dispersed distribution of labeled terminals was found in lamina VI (11%) (Table 2, Fig. 5b). Within lamina VI, labeling was more pronounced in the lateral region (8%) than the medial region (3%) (Table 2). The third strongest contralateral projection to the intermediate zone ended in lamina VIII (6%) (Fig. 5b). Comparatively fewer labeled boutons were found in the RMB (4%) and a small contingent of labeled terminals (less than 1%) were detected in contralateral lamina V (Table 2, Fig. 5b).

3.1.3. Ventral Horn Labeling

Lamina IX contained on average 12% of the total number of contralateral labeled boutons, representing the second strongest contralateral CSP from M1r (Table 2; Figs. 3c, e, f; 5b). Within lamina IX, the heaviest labeling was distributed in the dorsomedial quadrant (54%) followed by the dorsolateral quadrant (29%) then ventromedial quadrant (16%) (Fig. 6b). Sparse terminal labeling (1%) was noted in the ventrolateral quadrant (Fig. 6b).

3.1.4. Dorsal Horn Labeling

In all cases, terminal boutons from M1r were not detected with the stereology probe over the contralateral dorsal horn laminae (I-IV) (Table 2).

3.1.5. Segmental Organization

In the rostrocaudal dimension, the densest projection ended in C5 in all cases followed by a gradual decline in total bouton number from C6 to T1 (Fig. 7a). For example, when considering the average total number of boutons estimated for each segmental level, 33% of labeled boutons were found at C5, 26% at C6, 22% at C7, 12% at C8, and 8% was found at T1.

A more detailed level of investigation was carried out on segmental labeling within lamina VII and within lamina IX since these laminae received the strongest corticospinal input and lamina IX contains motoneurons. A stepwise segmental decline occurred in the CSP to lamina VII (Fig. 7c, see black bars). In terms of the segmental distribution of terminal labeling within the various subsectors of lamina VII, a dispersed pattern was found, identical to our overall analysis of lamina VII labeling noted above. For example, terminal labeling at all segmental levels (C5-T1) was dispersed in a relatively abundant manner over all lamina VII subsectors (Fig. 7c). Our segmental analysis of terminal labeling in lamina IX did not reveal a stepwise decline of boutons from C5 to T1. Instead, our analysis revealed a relatively consistent projection from C5 to C7, with far fewer boutons at C8 and T1 (Fig. 7e, see black bars). In terms of the lamina IX subsectors investigated, at C5 to C7 most terminals occupied the dorsomedial and dorsolateral quadrants (Fig. 7e).

3.2. Ipsilateral M1r Projection

3.2.1. Overall Laminar Organization of Terminal Labeling

Our quantitative stereological analysis demonstrated that the ipsilateral projection from M1 was primarily confined to the intermediate zone (laminae VIII and VII) with fewer terminals occurring in the ipsilateral midline region (lamina X) and ipsilateral ventral horn (Table 3, Fig. 5c).

Relative to the total number of labeled boutons estimated for the ipsilateral M1r projection, the highest percentage of labeled boutons was consistently found in lamina VIII (50%) followed by an average of 38% of labeled boutons in lamina VII (Fig. 5c). Only 8% of the average ipsilateral terminal labeling was found in motoneuron lamina IX, followed by an estimated 5% of terminals in the ipsilateral half of lamina X (Fig. 5c).

3.2.2. Intermediate Zone Labeling

Within ipsilateral lamina VII our analysis revealed that the most prominent labeling occurred in the ventromedial region (49%) (Fig. 6c). The second highest terminal distribution was found in the ventral ipsilateral sector (35%) followed by the dorsomedial subsector (11%) (Fig. 6c). The remainder of labeled boutons occupied the dorsolateral and ventrolateral regions of ipsilateral lamina VII.

3.2.3. Ventral Horn Labeling

As noted above, lamina IX labeling represented 8% of the total number of ipsilateral labeled boutons (Fig. 5c) but this projection was absent in case SDM57 (Table 3, Figs. 5c, 6d). In terms of topography, when present in the other 2 cases, labeling was located primarily within the ventromedial quadrant (Fig. 6d). In case SDM92, a comparatively strong projection was also found in the dorsomedial quadrant (Fig. 6d).

3.2.4. Dorsal Horn Labeling

The ipsilateral dorsal horn did not contain M1r terminal boutons (Table 3).

3.2.5. Segmental Organization

Our segmental analysis of the total ipsilateral projection showed the highest number of labeled boutons was located at C5 and C6, followed by a gradual decline of terminal labeling from spinal levels C7 to T1 (Fig. 7b). Our segmental analysis of total labeling within the various subsectors of lamina VII demonstrated a steady decline of labeling (Fig. 7d, see black bars) and this was paralleled by a steady decline in the relatively robust projection to the ventromedial and ventral subsectors (Fig. 7d, see green and blue bars respectively). Ipsilateral lamina IX labeling was primarily found at levels C5 and C6 (Fig. 7f). At both levels, labeling was located primarily within the dorsomedial and ventromedial regions of lamina IX (Fig. 7f, see blue and green bars respectively) with fewer terminals in the ventromedial region at inferior cervical spinal levels C7-T1.

4.0. DISCUSSION

The caudal region of the primary motor cortex (M1c) has attracted considerable attention because in some primates it harbors numerous CM projection neurons which provide the capacity for the performance of skilled use of the hand. However, far less is known about the contribution of the rostral region of the primary motor cortex (M1r) to cortical control of upper extremity movement. The present study represents the first effort to quantify the terminal distribution of the CSP from M1r to spinal levels C5-T1. Most of the M1r CSP terminated contralaterally (90%) (Fig. 5a). Our findings show the contralateral projection predominately targets laminae VII and to a lesser extent laminae IX, VIII and VI, with fewer terminals in laminae V and X and the RMB (Fig. 5b). The projection to lamina VII was by far the densest, being distributed in a relatively dispersed manner over all subsectors (Figs. 3, 6a). The M1r CSP to lamina IX chiefly innervated the dorsomedial quadrant, noted for harboring motoneuron somas innervating the proximal upper extremity, with relatively fewer terminals occupying the distal related dorsolateral quadrant and much less in the ventromedial quadrant (Figs. 3c, 3e, 3f, 6b).

The ipsilateral projection represented 10% of the average CSP with the primary target being lamina VIII, noted for harboring axial related motoneurons (Fig. 5c). This was followed by a slightly less dense projection to lamina VII, where the primary terminal target was the proximal related ventromedial subsectors (Fig. 6c).

The terminal distribution of the M1r CSP was found to represent a transitional CSP, when compared to the caudally adjacent M1c CSP, and rostrally adjoining LPMCd CSP. The anatomical findings supporting this “transitional” characterization are discussed below.

4.1. Comparative Analysis of Upper Extremity Corticospinal Terminations

Over the years our experiments, aimed to characterize the terminal distribution of the CSP from upper extremity frontal motor areas, have been designed with the intent that objective comparisons could eventually be made across the acquired terminal projection patterns. This has been achieved in part, by applying an identical experimental approach when injecting high-resolution anterograde dextran tract tracers into the cortical upper extremity motor representations. In these experiments, we placed 3 tracer injections (1.2μL total volume) spaced 1.5mm apart in a triangular pattern into the central region of an ICMS defined arm-related area, targeting subpial tracer involvement of cortical layers III-V. Following injection, all cases survived for a period of 32–34 days. To further standardize our experimental approach, we applied the same rigorous stereological design to quantify and estimate corticospinal terminal bouton numbers from C5 to T1.

For the discussion below, the estimated bouton numbers for the CSP from M1c hand/arm area were determined by averaging the findings from 3 M1c animal experiments (Morecraft et al., 2013), as were the estimated number of boutons for the M1r arm/hand CSP (Tables 2 and 3). Similarly, the M2/SMA arm area CSP findings represent an average of 4 animal experiments (McNeal et al., 2010, see control cases). Our LPMCd CSP estimate was obtained from one experimental animal (SDM57-FD). The SDM57-FD injection site was intentionally placed in the central region of the lateral premotor “arm/shoulder” region based upon comprehensive ICMS maps of the lateral premotor cortex (Godschalk et al., 1995; Kwan et al., 1978), and the epicortical stimulation maps of Woolsey and colleagues ((Woolsey et al., 1952); see Fig. 17a, b in Morecraft et al., 2019). Additional LPMC experimental cases will be noted when appropriate.

Our findings show that the terminal CSP patterns from these motor representations vary in their spinal termination, with some common or shared characteristics, as well as some unique or distinguishing characteristics. These occur in corticospinal laterality, total number of estimated corticospinal terminals as well as topographical and segmental distribution within lamina VII and motoneuron lamina IX.

4.1.1. Laterality

Our results show that a progressive stepwise shift in laterality occurs from the caudal part of the precentral cortex (M1c), to the most rostral part of premotor cortex. For example, the M1c CSP was found to be 98% contralateral (Morecraft et al., 2013) (Fig. 8a). Progressing rostrally, the present results show the M1r CSP is on average 90% contralateral, and the CSP from the adjoining arm/shoulder area of LPMCd is 79% contralateral (Morecraft et al., 2019; see case SDM57-FD). This gradual decrease in laterality continues when considering the CSP from the most rostral part of LPMCd is 72–73% contralateral (Morecraft et al., 2019; see cases SDM61-FD and SDM54-BDA) (Fig. 8a). Therefore, in terms of laterality the M1r CSP represents a “transitional” CSP amongst the upper extremity motor representations positioned on the lateral cortical surface (Fig. 8a).

Figure 8.

Transitional nature of the corticospinal projection originating frontal motor areas controlling muscles of the upper limb. (a) Percentage of total boutons from lateral cortical motor areas to all spinal laminae of C5-T1 (blue bars are the percentage for the contralateral projection, red bars are the percentage for ipsilateral projection). The numbers above the bars in (a) are the percentages of the contralateral and ipsilateral projections. (b) Estimated number of boutons from frontal motor areas in the CSP to all contralateral spinal laminae of C5-T1. (c) Estimated number boutons from frontal motor areas in the CSP to contralateral lamina VII of C5-T1. (d) Estimated number of boutons in the CSP to contralateral lamina IX of C5-T1. Abbreviations: LPMCd(r), most rostral portion of the dorsal lateral premotor cortex (cases SDM61-FD, SDM54-BDA); LPMCd, arm/shoulder part of dorsal lateral premotor cortex (case SDM57-FD); LPMCv, ventral (periarcuate) portion of the lateral premotor cortex (case SDM72-FD); M1c, caudal portion of primary motor cortex lining the anterior wall of the central sulcus; M1r, rostral portion of the primary motor cortex on the gyral convexity; M2, supplementary motor cortex.

Further examination of the CSP results from each individual M1r case reveals a progressive “intrinsic” trend, or gradient of laterality change, that compliments the overall trend summarized above (Fig. 8a). From caudal to rostral M1r laterality gradually changed from 94.6% contralaterally for the most caudal injection site (SDM57), to 91% (SDM89) then 82.9% for the most rostral injection site (SDM91) (Figs. 1, 5a).

4.1.2. Terminal Density

In terms of estimated total bouton number and overall terminal density, the strongest/densest contralateral CSP to the cervical enlargement (C5-T1) clearly originates from the hand/arm area of M1c (Morecraft et al., 2013- Table 2, average = 199,214 boutons) (Fig. 8b). This is followed by the contralateral terminal CSP from the M2/SMC arm area (McNeal et al., 2010- Table 2, average = 75,853 boutons), M1r arm/hand area (Table 1, average = 44,003), LPMCd arm/shoulder area (Morecraft et al., 2019- Table 1, SDM57-FD = 35,469 boutons) and finally the LPMCv periarcuate region (Morecraft et al., 2019- Table 4, SDM72-FD = 6,162 boutons). This hierarchy in the stepwise decline of estimated boutons occurred within contralateral lamina VII (M1c = 118,320; M2/SMC = 47,320; M1r = 28,529; LPMCd = 22,328; LPMCv =5,776) (Fig. 8c) and within contralateral lamina IX (M1c = 36,464; M2/SMA = 11,277; M1r = 5,105; LPMCd = 1,218; LPMCv = 0) (Fig. 8d). Thus, when considering the total number of contralateral boutons for the lateral motor representations (M1c, M1r and LPMCd), M1r represents a transitional region between the caudally adjoining M1c, and rostrally adjoining LPMCd (Fig. 8B).

These observations closely parallel the retrograde transport findings of Galea and Darian-Smith (Galea & Darian-Smith, 1994) who found the largest number of contralateral corticospinal neurons reside in M1 following injections of retrograde tracer into the cervical enlargement of the spinal cord. They reported that the second highest number of contralateral corticospinal neurons were located in M2/SMC (identified as area F3/SMA/mesial area 6aα) followed by comparatively fewer corticospinal projection neurons located in LPMCd (identified as area F2/dorsolateral area 6aα) and fewest labeled neurons in the region of the arcuate spur (identified as the post-arcuate area). A similar trend of retrograde labeling following cervical spinal cord injections was found by Dum and Strick (Dum & Strick, 1991).

Unlike the progressive “intrinsic trend” found for M1r laterality, we did not find a progressive increase in terminal density from rostral to caudal regions within M1r, which would be expected given the overall trend of the lateral upper extremity motor representations (Fig. 8b). Indeed, cases SDM89 and SDM92 were rostral in M1r and gave rise to the highest number of terminations (Fig. 5a, b; Table 2), with less terminations estimated for caudal case SDM57. It is possible that this may be due to the wide range of ages, with cases SDM89 and SDM92 being very young (1 and 1.2 years respectively) compared to the much older case SDM57 (18 years). Another possibility is one of the deposited dextran injections (of the 3 placed in a triangular pattern) in case SDM57 did not reach layer V for spinal cord transport, as layer V is the exclusive cortical layer harboring cell bodies giving rise to CSP axons (Kuypers, 1981; Martin, 2005). However, we did not have a complete set of serial sections through the injection site to evaluate this possibility. Despite this, we are inclined to interpret the location of the SDM57 injection site as being adjacent to M1c based upon the cortical position of the histologically reconstructed injection site (Fig. 1), the percent laterality of the SDM57 CSP as discussed above, as well as the topography of the SDM57 lamina VII and IX CSP discussed below.

4.1.3. Topographical and Segmental Distribution within Contralateral Lamina VII

The primary terminal target of the contralateral corticospinal projection from all frontomotor areas (M1c, M1r, LPMCd, LPMCv and M2) was lamina VII of the intermediate zone. For the M1c, M1r and LPMCd CSP experiments, lamina VII was divided into 5 subsectors. This analysis demonstrated the contralateral lamina VII CSP from these motor regions differ in terms of their overall termination pattern, with the intervening M1c CSP having some common characteristics of both M1c and LPMCd.

In our previous study, we had found the contralateral lamina VII projection from the M1c hand/arm area primarily targeted the dorsolateral subsectors (see Fig. 10A in Morecraft et al., 2013) in agreement with Kuypers earlier findings (Kuypers & Brinkman, 1970). Kuypers recognized the dorsal and lateral parts lamina VII to be affiliated with the distal related “lateral motor system” because propriospinal neurons in these regions give rise to ipsilateral short-range projections (Kuypers, 1982; Molenaar, 1978; Sterling & Kuypers, 1968). Furthermore, these short-range propriospinal neurons preferentially innervate lamina IX motoneurons of the distal upper extremity muscles (Kuypers, 1982; Sterling & Kuypers, 1968).

In contrast to the M1c findings, we previously found that the arm/shoulder LPMCd CSP heavily targeted the ventromedial sectors of lamina VII bilaterally (see Fig. 7a in Morecraft et al., 2019), also in agreement with Kuypers (Kuypers & Brinkman, 1970). Kuypers recognized the ventromedial region of lamina VII (including adjacent lamina VIII) to be key components of the proximal related “medial motor system” because they harbor propriospinal neurons with long-range bilateral projections (Kuypers, 1982; Matsushita et al., 1979; Molenaar & Kuypers, 1978). Furthermore, the short propriospinal neurons located in the ventral parts of lamina VII preferentially innervate lamina IX motoneurons of the proximal extremity and girdle muscles, including lamina VIII motoneurons of the axial muscles (Kuypers, 1982; Sterling & Kuypers, 1968). Additional experimental support considered for interpreting the ventromedial CSP terminal site was provided by Lawrence and Kuypers who demonstrated interruption of the ventromedial brainstem pathway to the cord, in the absence of corticospinal inputs (achieved by bilateral pyramidotomy), resulted in severely impaired axial (postural) and proximal upper extremity movements (Lawrence & Kuypers, 1968)(see (Lemon et al., 2012) for overview).

In the present study, we found the contralateral M1r CSP to be consistently distributed over all 5 subsectors of lamina VII at all segmental levels (Figs. 6a see mean, 7c), thus targeting both the distal and proximal related regions of lamina VII. However, when considering the distribution of lamina VII terminations of the individual M1r cases, a clear picture of a transitional termination pattern emerges. The most caudal M1r injection site (SDM57) preferentially terminated in the dorsolateral region of lamina VII (Fig. 6a, note the DM, DL and VL columns) which is the lamina VII distribution characterizing the M1c CSP (see Fig. 10A in Morecraft et al., 2013). In contrast, the most rostral M1r injection site (SDM92) produced dense terminations in the ventromedial region (Fig. 6, note the VM, VL, and V columns), which is the characteristic lamina VII distribution of the adjacent LPMCd arm/shoulder area (see Fig. 7A, SDM57 in Morecraft et al., 2019). Neither the “dorsolateral” or “ventromedial” pattern characterized intervening case SDM 89 (Fig. 6a).

4.1.4. Topographical and Segmental Distribution within Contralateral Lamina IX

All three lateral motor representations (M1c, M1r and LPMCd) gave rise to a contralateral projection to the dorsomedial quadrant of lamina IX at C5 to C7 (M1r- Fig. 6b, 7e; M1c- see Figs. 10B, 11E in Morecraft et al., 2013; LPMCd - see Figs. 8 and 10c in Morecraft et al., 2019). The dorsomedial quadrant at these spinal levels harbors proximal limb motoneurons, including somas innervating the biceps brachialis (Jenny and Inukai, 1983) which produce flexion at the shoulder and elbow (Fig. 9b, c). Consistent with this interpretation are the findings of Reed (Reed, 1940). Following transection of the musculocutaneous nerve as it emerged from the brachial plexus in the axilla, Reed reported degenerating motoneurons in the “medial part of the dorsolateral group” at C5-C7, with most degenerating neurons occurring at C6. The anatomical region described by Reed corresponds to the dorsomedial quadrant of lamina IX.

Figure 9.

Summary diagram showing the general topography of the M1r CSP found in the present study (b) compared to the topography of the M1c CSP (a) and LPMCd CSP (c) found in our previous studies (Morecraft et al., 2013, 2019). To illustrate the preeminent and distinguishing structural features of each CSP, the M1c CSP is shown at distal spinal levels C8 and T1, whereas the M1r and LPMCd CSP are shown at proximal spinal levels C5 to C7. These cervical levels also reflect the motor sequalae that accompany caudal motor cortex injury (a: distal deficits), and rostral motor cortex injury (c; proximal/axial deficits). Each terminal pattern is also shown in relation to Kuypers “lateral motor system” (blue: see diagonal hatching and circles on the horizontal spinal cord section in Fig. 9 of Kuypers, 1982) and “medial motor system” (red: see the light stippling on the horizontal spinal cord section in Fig. 9 of Kuypers, 1982). Light blue identifies the motoneuron component of Kuypers lateral motor system (lamina IX) and light red the motoneuron component of Kuypers “medial motor system” (lamina VIII). Panel (a) illustrates the predominately contralateral (98%) M1c CSP to dorsolateral sectors of lamina VII and widespread lamina IX projection at C8-T1. This CSP represents a central component of Kuypers distal-related “lateral motor system”. In contrast panel (c) shows the bilateral (79%) LPMCd CSP to the ventromedial region of lamina VII and the limited projection to lamina IX at C5 to C7. This CSP represents a key component of Kuypers proximal-related “medial motor system”. As found in the present study, the M1r CSP in panel (b) shows terminal characteristics of both the lateral and medial motor systems illustrating the transitional nature of the M1r CSP. For comparative purposes, if the M1c CSP were shown at spinal levels C5-C7, the illustrated topography would be exactly the same as shown in panel (a), with the exception that only the dorsolateral and dorsomedial quadrants of lamina IX would be shaded in light blue (as in panel (b) for M1r). On the cortex (top) in panels (a) and (b) the central sulcus has been “opened” to show the cortex lining the anterior bank of the sulcus (shaded in light gray). Differences in terminal density of each descending CSP pathway are correlated to line thickness, with denser terminal projections represented by increased line thickness and arrowhead size.

In addition to the M1r and M1c CSP to the dorsomedial quadrant noted above, both motor representations innervated the dorsolateral quadrant at C5-C7. (M1r- Fig. 7e; M1c- see Figs. 11E and 12B in Morecraft et al., 2013). Motoneuron somas known to reside in the dorsolateral quadrant at C5 to C7 innervate the flexor carpi radialis (FCR) and extensor carpi radialis (ECR) (Jenny & Inukai, 1983), both acting distally at the wrist joint. Degenerating motoneurons occur in this same location following proximal transection of the median nerve (Reed, 1940). The location of wrist extensor motoneurons being in the dorsolateral region of lamina IX at C5-C7 would also be consistent with the topographic distribution of motoneurons forming the ulnar nerve, as determined after injecting a fluorescent retrograde tracer directly into the ulnar nerve above the elbow (Chiken et al., 2001).

Yet, there was an important difference between M1r and M1c CSP with respect to the segmental and topographical distribution of estimated boutons within lamina IX. For M1r, we found a general decline in total number of contralateral lamina IX boutons from C5 to T1 (Fig. 7e). In contrast, not only did the M1c CSP increase in bouton number from C5 to C8/T1, this projection was widely distributed among all lamina IX quadrants at C8/T1 (Fig. 9a; see Fig 11E and 12 in Morecraft et al., 2013). This observation shows dense, and potential widespread terminations from M1c on motoneuron somas (and their proximal dendrites) innervating flexors, extensors, abductors and adductors acting on the digits, hand and wrist (Jenny & Inukai, 1983). This interpretation would be aligned with fluorescent retrograde multi-labeling observations showing extensive and widespread intermingling of labeled motoneurons in lamina IX at C8 and T1 following isolated injections of different colored tracers into the median, ulnar and radial nerves just above the elbow joint in the same monkey (Chiken et al., 2001).

In summary, all three lateral upper extremity motor representations (M1c, M1r and LPMCd) projected to the dorsomedial quadrant of lamina IX at C5-C7 (Fig. 9b, c). Both M1r and M1c additionally sent a substantial projection to dorsolateral quadrant at C5-C7 (M1r - see Fig. 9b; M1c - see Fig. 9 legend). M1c was the only CSP to heavily terminate at distal spinal levels C8 and T1, and this included widespread terminations within all lamina IX quadrants (Fig. 9a).

4.2. Cortico-motoneuronal Projections from M1r versus M1c: Examining the Anatomical and Neurophysiological Evidence.

Our study complements several other approaches that have been used to compare the characteristics of M1r vs M1c and their respective corticospinal and CM projections. Early on, a subdivision of M1 into M1c and M1r was suggested on the basis of differences in somatosensory input into caudal (cutaneous) vs rostral (proprioceptive) divisions of macaque motor cortex (Lemon, 1981; Strick & Kim, 1978; Strick & Preston, 1982; Tanji & Wise, 1981), and on the basis of neurotransmitter distribution and cytoarchitecture in human M1 (Geyer et al., 1996). It was the use of viral retrograde transneuronal labelling by Rathelot & Strick (Rathelot & Strick, 2006, 2009) that firmly established the subdivision, based upon the distribution of CM neurons projecting to upper limb muscles. These investigations involved second-order retrograde transneuronal labelling of CM cells obtained after virus injection into single arm, hand and digit muscles in the macaque monkey. The timing of these experiments is critical, since the animal has to be sacrificed when maximum second-order labelling has been achieved (i.e., in layer V CM neurons) but before the virus spreads to give third-order labelling in connected neurons (e.g., non-CM pyramidal neurons in layers III and V (Strick et al., 2021).

CM neurons were mostly found in M1c, with heavily overlapping representations of different intrinsic and extrinsic digit muscles (Rathelot & Strick, 2006), and a partial overlap between distal and proximal arm muscles (Rathelot & Strick, 2009). In contrast, CM cell distribution within M1r was sparse: for digit muscles around 94% of CM cells in M1 were located in M1c, with only 6% in M1r. For the proximal arm muscles, the figures were very similar with only 6–8% in M1r. Individual CM neurons have divergent projections (Buys et al., 1986; Fetz & Cheney, 1980; McKiernan et al., 1998), so that the retrogradely-labelled neurons could innervate other muscles in addition to the one injected with virus.

The anterograde labelling approach used here also demonstrated a clear contrast between the high density of labelled corticospinal boutons in contralateral cervical laminae VII and IX from M1c (an average total of 154,784 boutons across 3 cases; Morecraft et al 2013, Table 2) versus much lower numbers from M1r (33,634 boutons; current study). Combining the results from both these studies, if we add all the boutons found in segments C5-T1 in the contralateral lamina IX from M1c and M1r (41,569), then 88% of these originate from M1c and only 12% from M1r. Interestingly, the difference is somewhat greater for lamina IX in segments C8 and T1, which harbor most of the hand and digit motoneurons (Jenny & Inukai, 1983). In these segments 95% originate from M1c and 5% from M1r. These differences are very similar to those for the transneuronal method (94% vs 6%).

However, it is difficult to compare the results obtained with the anterograde and retrograde approaches. We do not know how anterogradely labelled corticospinal boutons are distributed across different motoneurons belonging to different muscles, so differences may be expected when comparing with retrograde labelling of CM neurons from virus injections in a single muscle. This is particularly the case since we know that the CM input varies considerably across different motor units within a muscle, being generally stronger for low threshold motor units (Awiszus & Feistner, 1994; Gandevia & Rothwell, 1987; Lemon & Mantel, 1989; Mantel & Lemon, 1987). The total CM input also varies across muscles (Clough et al., 1968; Porter, 1993; Witham et al., 2016). We don’t know whether all labelled boutons within lamina IX are on alpha motoneurons, or how many CM cells converge to innervate each motoneuron. The dendrites of alpha motoneurons are very extensive, and retrograde labelling of CM cells in M1 may include virus transfer through synapses on distal dendrites of innervated motoneurons. Many of these would be located outside lamina IX, in lamina VII (Lawrence et al., 1985; Sinopoulou et al., 2022), which lamina receives an even larger projection from M1 than does lamina IX (Table 2; Morecraft et al 2013).

Together, the overall anatomical results suggest that there are likely to be some limited CM projections from M1r to motoneurons in the contralateral cervical cord. A similar conclusion was reached by Witham et al. (2016), who compared, in anaesthetized macaques, the neurophysiology of CM projections from M1c and M1r. They delivered single intracortical stimuli to these subdivisions and evoked excitatory postsynaptic potentials (EPSPs) in 135 spinal motoneurons innervating a variety of arm and digit muscles. These stimuli can excite CM cells directly, but they also give rise to high-frequency discharge of the corticospinal system (the so-called D (direct) and I (indirect waves), with complex effects on target motoneurons (Lemon et al., 2002; Porter, 1993; Witham et al., 2016).

Witham et al (2016) found that some short-latency EPSPs could be unequivocally defined as monosynaptic (i.e., CM), since they began only a short time (< 1.0 ms) after the arrival of the earliest descending corticospinal volley (D-wave) at the relevant segment. These monosynaptic EPSPs were all evoked from M1c, as might be expected from the anatomical studies. However, these EPSPs were very much in the minority (27/135): a far greater number of motoneurons (108) showed longer-latency EPSPs which were considered to be either monosynaptic responses evoked by more slowly conducting corticospinal volleys, or mediated by oligosynaptic pathways. Long-latency monosynaptic effects occurred with similar incidence from both M1c and M1r, although they were of larger amplitude from M1c than M1r. Again, the overall result suggests a much greater direct excitatory influence over upper limb motoneurons from M1c than from M1r. Sparser CM input to lamina IX and to distal dendrites in lamina VII from M1r might explain the weaker influence of this subdivision: similar features have been suggested to explain the weak CM input to hand motoneurons in the New World squirrel monkey (Maier et al., 1997).

The findings of Witham et al (2016) emphasized the contribution of CM cells with relatively slowly conducting axons, evoking EPSPs with latencies as much as 3 or 4 times longer than the fastest CM-EPSPs. In keeping with this result, Rathelot & Strick (2006) found that retrogradely labelled CM neurons had a wide range of soma sizes, indicating that the CM projection originated from both large neurons with fast conducting axons, and smaller neurons with slow axons. Because slower axons are so much more numerous than fast ones (Firmin et al., 2014), these slow effects are likely to dominate the CM influence over the upper limb.

Another neurophysiological approach that has been used to study the CM system is the spike-triggered averaging (STA) technique, which identifies CM neurons in the awake macaque by averaging EMG activity in relation to the spontaneous discharges of single M1 neurons during a trained movement (Fetz & Cheney, 1980; Porter, 1993; Schieber & Rivlis, 2005). Most published studies demonstrate CM cells identified with this approach to be located in macaque M1c (anterior bank of the central sulcus) (Griffin et al., 2015; Lemon et al., 1986; McKiernan et al., 1998; Schieber & Rivlis, 2005) but there also appear to be a few CM cells located in the convexity of the gyrus (M1r) (see Fig. 4B-E in Lemon 1988; McKiernan et al 1998;(Schieber & Rivlis, 2005). However, no systematic study has yet been carried out.

In humans, although non-invasive stimulation (TMS, TES) of the motor cortex has provided direct evidence of CM connections in the human corticospinal system (e.g. (Day et al., 1989; de Noordhout et al., 1999), the relatively unfocused nature of the induced excitation makes it difficult to distinguish activation of M1c versus M1r. Direct electrical stimulation of the motor cortical tissue during intra-operative surgery uses a monopolar probe and a brief train of high-frequency pulses provides more focused stimulation. (Vigano et al., 2019) showed that, with this approach, there was a caudal to rostral gradient across the precentral gyrus, such that the motor evoked responses from contralateral hand and digit muscles had lower thresholds, larger amplitudes and shorter latencies from the caudal sector than from the rostral sector. These results might reflect an M1c-M1r divide. The hand region of M1c (M1p in human nomenclature; Geyer et al 1996) is largely buried deep in the anterior bank of the central sulcus (Glasser et al., 2016) and that may influence its susceptibility to stimulation of the precentral cortical surface.

It is of course important to recognize that much of the excitatory drive to spinal motoneurons come from non-CM inputs (Lemon, 2008). For example, the reticulospinal system has been identified as a major source of this drive (Baker 2011; Tapia et al 2022), particularly for the generation of high forces (Glover & Baker, 2022) and for strength training (Glover & Baker, 2020). In this respect it is important that the neurons giving rise to the reticulospinal tract receive extensive convergence from motor areas of the cortex, including M1r (Fisher et al., 2021; Fregosi et al., 2017; Keizer & Kuypers, 1989). Oligosynaptic projections to forelimb motoneurons are also evidenced in the stimulation study of Witham et al., (2016) and the finding of a heavy CSP to lamina VII in the C5-T1 segments (Table 2, Fig. 5), as shown in the present study and by Morecraft et al., (2013).

4.3. Clinical Considerations of a Transitional Lateral Upper Extremity CSP

The findings presented on the terminal organization of the M1r CSP, along with our previous findings on CSP terminal organization from adjoining lateral motor representations M1c and LPMCd, appear to correlate with the clinical motor sequelae that accompany caudal versus rostral injury to the lateral precentral/premotor region. More to the point, when considering the progressive nature and gradual shift of all 3 lateral corticospinal projection patterns, the distal to proximal CSP gradient appears to reflect the distal to proximal motor deficits that characterize caudal and rostral lesions on the lateral frontal surface.

Although representing less than 1% of all strokes (Finkelsteyn et al., 2019), precentral lesions localized to cortex lining the anterior wall of the central sulcus (commonly referred to the “motor hand area” or “hand knob area”) can result in isolated weakness/paralysis of the fingers (e.g., (Schieber, 1999); (Gass et al., 2001); (Takahashi et al., 2002); for review see Finkelsteyn et al., 2019). Our findings of the M1c CSP, which primarily originates from cortex lining the anterior bank of the central sulcus, would firmly predict the clinical outcome of hand/digit dysfunction (Morecraft et al., 2013). As noted, we found the CSP from M1c to motoneuron lamina IX represents the second highest proportion of contralateral boutons (18%), and the lamina IX projection gradually increases in terminal density from C5 to C8/T1. Furthermore, at spinal levels C8 and T1, we found CSP terminals to be evenly dispersed in all lamina IX quadrants, indicating widespread innervation on motoneuron somas and proximal dendrites which in turn innervate flexors, extensors, abductors and adductors acting on the digits, hand and wrist (Jenny and Inukai, 1983) as noted above (Fig. 9a). Thus, injury affecting the cortex forming the anterior bank of the central sulcus would primarily affect movements of the hand and motor skills requiring fractionated finger movements (Khanna et al., 2021).

In contrast, clinical evidence has shown patients with unilateral premotor cortex injury present primarily with weakness of the contralateral shoulder, mainly affecting muscles involved with abduction and elevation of the arm (Freund, 1987; Freund & Hummelsheim, 1984, 1985), 1985). This clinical “premotor syndrome” is additionally characterized by disturbed postural support (Freund, 1990), and EMG analyses show considerable preactivation delays occur in proximal arm movement, hindering normal proximal-distal synergies of muscle action (Freund & Hummelsheim, 1985). A more recent, and complementary study has shown that virtual lesions (via TMS) to dorsocaudal premotor cortex (PMdc) in human subjects delay the recruitment of the proximal muscles involved in the lifting phase during grasping and lifting movements (Davare et al., 2006). Although such deficits are likely attributed to the compromise of many neural circuits including cortico-cortical and corticostriate networks to mention a few, the occurrence of proximal and postural neurological deficits following unilateral dorsal premotor damage disclose a strong parallel with what we have reported for the terminal corticospinal projection from the arm/shoulder region of premotor cortex (LPMCd), and our interpretations and conclusions based upon Kuypers “medial motor system” model of the descending motor pathways (Morecraft et al., 2019). As we have pointed out, the LPMCd CSP is optimally organized to support proximal (shoulder and elbow) upper extremity movements including axial movements as determined by multiple intrinsic patterns of terminal labeling, including the topographical and segmental distribution of the terminal CSP to laminae VII, VIII and IX (see above, and Introduction) (Fig. 9c).

Our new findings on the M1r CSP to laminae VII and IX would predict that lesions affecting cortex located between the anterior bank of the central sulcus and premotor cortex would adversely affect both distal and proximal spinal mechanisms (Fig. 9b). What evidence would support this supposition? First, as noted above, small, localized lesions of the hand knob area can result in isolated hand/digit paresis. However, patients with a hand knob area lesion that concomitantly present with more proximal arm deficits often have cortical injury extending beyond the hand knob area (e.g., Schieber, 1999, cases 3 and 7; Gass et al., cases 1 and 3; Finkelsteyn et al., 2019). Second, the prediction of mixed proximal and distal arm deficits following M1r injury would relate to a somatotopic gradient within M1 as indicated in NHP electrophysiological mapping studies. This work has shown a central core of distal movements (wrist, digit and intrinsic hand) located in caudal M1 (Kwan et al., 1978; Park et al., 2004; Park et al., 2001). The distal core is surrounded by a belt of cortex mediating distal and proximal forelimb movements, which in turn is surrounded by a belt of cortex on the central sulcus convexity mediating proximal (elbow and shoulder) movements. Third, epicortical stimulation of the human precentral gyrus shows a pattern of intermingled distal and proximal joint movements on the gyral surface (Catani, 2017; Penfield, 1937). The rostral part of the precentral gyrus mapped by Penfield and Boldrey might be equivalent to M1r in the non-human primate, as a major portion of the human hand motor area (M1c) is buried in the depths of the central sulcus (Glasser et al., 2016; Ostergard & Miller, 2019).

4.4. Summary and Conclusions

We show the terminal distribution of the M1r CSP ends primarily in contralateral lamina VII, with fewer terminations ending in neighboring laminae of the intermediate zone and lamina IX of the anterior horn. When comparing the terminal CSP patterns to laminae VII and IX across M1c, M1r and LPMCd, the M1r CSP appears to represent a “transitional” CSP, positioned between the distal related M1c CSP, and proximal/axial related LPMCd CSP. These findings further substantiate Kuypers (1982) conceptual view of “lateral” and “medial” descending motor systems in the primate central nervous system (Fig. 9). We consider the M1r CSP in the light of neurophysiological and neuroanatomical approaches used to compare the CSP characteristics of M1r versus M1c. We conclude that most CM neurons are within M1c, with a limited number occupying M1r; this latter conclusion is supported by our finding terminations from M1r in motoneuron lamina IX. Finally, we point to the clinical evidence which suggests the distinct changes in motor sequelae accompanying caudal and rostral lesions on the lateral frontal surface are closely linked to the progressive nature and gradual shift of all 3 (M1c, M1r and LPMCd) corticospinal projection patterns. With the M1c CSP pattern corresponding to the dominant distal upper extremity deficits accompanying caudal precentral (hand knob) injury, and the LPMCd CSP pattern corresponding to the dominant proximal upper extremity and axial deficits occurring after premotor injury. We advance the suggestion that our new findings for the M1r CSP would predict a mix of proximal and distal deficits following rostral precentral injury but would expect good recovery of motor deficits due to sparing of M1c and LPMCd.

COMMON ABBREVIATIONS:

CM

corticomotoneuronal

FD

fluorescein dextran

LPMC

lateral premotor cortex

LPMCd

dorsolateral premotor cortex

LPMCv

ventrolateral premotor cortex

LYD

lucifer yellow dextran

M1

primary motor cortex

M1c

caudal primary motor cortex

M1r

rostral primary motor cortex

Data Availability Statement:

All data for this study have been included in the main document and its supporting materials.