Corticospinal Neurons Research Articles

Muscle-specific modulation of indirect inputs to primary motor cortex during action observation.

Single-pulse transcranial magnetic stimulation (spTMS) studies report that movement observation facilitates corticospinal excitability in primary motor cortex (M1) in a muscle-specific manner. However, motor evoked potentials (MEPs) elicited by spTMS are known to reflect the summation of several descending volleys in corticospinal neurons which are evoked via mono- and polysynaptic inputs (so-called indirect waves or I-waves). It is unclear which of these components contribute to the muscle-specific modulation of M1 during action observation. The interactions between different I-waves are reflected in the facilitatory peaks elicited with a short-intracortical facilitation (SICF) protocol when two pulses are sent to M1 at precise intervals (i.e., 1.3, 2.5 or 4.1ms). Here, we explored the modulation of early and late SICF peaks during action observation by measuring highly specific MEP amplitude changes measured in two muscles (index, FDI and little finger, ADM) while participants observed two different actions (precision and whole-hand grip). Our results demonstrate that both early (1.3ms) and late (2.5 and 4.1ms) SICF peaks are modulated in the context of movement observation. However, only the second peak (ISI 2.5ms) was significantly associated with the muscle-specific modulation of corticospinal excitability as measured with spTMS. This late SICF peak is believed to reflect the activity cortico-cortical pathways involved in the facilitation of muscle-specific representations in M1. Thus, our findings suggest that movement observation leads to widespread activation of different neural circuits within M1, including those mediating cortico-cortical communication.

Growth-Promoting Treatment Screening for Corticospinal Neurons in Mouse and Man

Neurons of the central nervous system (CNS) that project long axons into the spinal cord have a poor axon regenerative capacity compared to neurons of the peripheral nervous system. The corticospinal tract (CST) is particularly notorious for its poor regeneration. Because of this, traumatic spinal cord injury (SCI) is a devastating condition that remains as yet uncured. Based on our recent observations that direct neuronal interleukin-4 (IL-4) signaling leads to repair of axonal swellings and beneficial effects in neuroinflammation, we hypothesized that IL-4 acts directly on the CST. Here, we developed a tissue culture model for CST regeneration and found that IL-4 promoted new growth cone formation after axon transection. Most importantly, IL-4 directly increased the regenerative capacity of both murine and human CST axons, which corroborates its regenerative effects in CNS damage. Overall, these findings serve as proof-of-concept that our CST regeneration model is suitable for fast screening of new treatments for SCI.

Evidence that distinct human primary motor cortex circuits control discrete and rhythmic movements.

Discrete and rhythmic dynamics are inherent components of (human) movements. We provide evidence that distinct human motor cortex circuits contribute to discrete and rhythmic movements. Excitability of supragranular layer circuits of the human motor cortex was higher during discrete movements than during rhythmic movements. Conversely, more complex corticospinal circuits showed higher excitability during rhythmic movements than during discrete movements. No task-specific differences existed for corticospinal output neurons at infragranular layers. The excitability differences were found to be time(phase)-specific and could not be explained by the kinematic properties of the movements. The same task-specific differences were found between the last cycle of a rhythmic movement period and ongoing rhythmic movements. Human actions entail discrete and rhythmic movements (DM and RM, respectively). Recent insights from human and animal studies indicate different neural control mechanisms for DM and RM, emphasizing the intrinsic nature of the task. However, how distinct human motor cortex circuits contribute to these movements remains largely unknown. In the present study, we tested distinct primary motor cortex and corticospinal circuits and proposed that they show differential excitability between DM and RM. Human subjects performed either 1) DM or 2) RM using their right wrist. We applied an advanced electrophysiological approach involving transcranial magnetic stimulation and peripheral nerve stimulation to test the excitability of the neural circuits. Probing was performed at different movement phases: movement initiation (MI, 20ms after EMG onset) and movement execution (ME, 200ms after EMG onset) of the wrist flexion. At MI, excitability at supragranular layers was significantly higher in DM than in RM. Conversely, excitability of more complex corticospinal circuits was significantly lower in DM than RM at ME. No task-specific differences were found for direct corticospinal output neurons at infragranular layers. The neural differences could not be explained by the kinematic properties of the movements and also existed between ongoing RM and the last cycle of RM. Our results therefore strengthen the hypothesis that different neural control mechanisms engage in DM and RM.

The Timing and Extent of Motor Neuron Vulnerability in ALS Correlates with Accumulation of Misfolded SOD1 Protein in the Cortex and in the Spinal Cord.

Understanding the cellular and molecular basis of selective vulnerability has been challenging, especially for motor neuron diseases. Developing drugs that improve the health of neurons that display selective vulnerability relies on in vivo cell-based models and quantitative readout measures that translate to patient outcome. We initially developed and characterized UCHL1-eGFP mice, in which motor neurons are labeled with eGFP that is stable and long-lasting. By crossing UCHL1-eGFP to amyotrophic lateral sclerosis (ALS) disease models, we generated ALS mouse models with fluorescently labeled motor neurons. Their examination over time began to reveal the cellular basis of selective vulnerability even within the related motor neuron pools. Accumulation of misfolded SOD1 protein both in the corticospinal and spinal motor neurons over time correlated with the timing and extent of degeneration. This further proved simultaneous degeneration of both upper and lower motor neurons, and the requirement to consider both upper and lower motor neuron populations in drug discovery efforts. Demonstration of the direct correlation between misfolded SOD1 accumulation and motor neuron degeneration in both cortex and spinal cord is important for building cell-based assays in vivo. Our report sets the stage for shifting focus from mice to diseased neurons for drug discovery efforts, especially for motor neuron diseases.

L1CAM Is a Marker for Enriching Corticospinal Motor Neurons in the Developing Brain

The cerebral cortical tissue of murine embryo and pluripotent stem cell-derived neurons can survive in the adult brain and extend axons to the spinal cord. These features suggest that cell transplantation can be a strategy to reconstruct the corticospinal tract (CST). It is unknown, however, which cell population makes for safe and effective donor cells. To address this issue, we grafted the cerebral cortex of E14.5 mouse to the brain of adult mice and found that the cells in the graft extending axons along the CST expressed CTIP2. By using CTIP2:GFP knock-in mouse embryonic stem cells (mESCs), we identified L1CAM as a cell surface marker to enrich CTIP2+ cells. We sorted L1CAM+ cells from E14.5 mouse brain and confirmed that they extended a larger number of axons along the CST compared to L1CAM− cells. Our results suggest that sorting L1CAM+ cells from the embryonic cerebral cortex enriches subcortical projection neurons to reconstruct the CST.

Regeneration of Corticospinal Axons into Neural Progenitor Cell Grafts After Spinal Cord Injury.

Spinal cord injuries leave patients with lifelong paralysis. To date, there are no therapies that promote the critical step required for the recovery of voluntary motor function: corticospinal axon regeneration. Spinal cord-derived neural progenitor cell (NPC) grafts integrate into the injured host spinal cord, enable robust corticospinal axon regeneration, and restore forelimb function following spinal cord injury in rodents. Consequently, engineered stem cell differentiation and transplantation techniques harbor promising potential for the design and implementation of therapies promoting corticospinal axon regeneration. However, in order to optimize the outcome of clinical trials, it is critical to fully understand the cellular and molecular mechanisms underlying this regeneration. Our recent study highlights the unexpected intrinsic potential of corticospinal neurons to regenerate and allows us to investigate new hypotheses exploiting this newly discovered potential.

Priming Effects of Water Immersion on Paired Associative Stimulation-Induced Neural Plasticity in the Primary Motor Cortex.

We aimed to verify whether indirect-wave (I-wave) recruitment and cortical inhibition can regulate or predict the plastic response to paired associative stimulation with an inter-stimulus interval of 25 ms (PAS25), and also whether water immersion (WI) can facilitate the subsequent PAS25-induced plasticity. To address the first question, we applied transcranial magnetic stimulation (TMS) to the M1 hand area, while alternating the direction of the induced current between posterior-to-anterior and anterior-to-posterior to activate two independent synaptic inputs to the corticospinal neurons. Moreover, we used a paired stimulation paradigm to evaluate the short-latency afferent inhibition (SAI) and short-interval intracortical inhibition (SICI). To address the second question, we examined the motor evoked potential (MEP) amplitudes before and after PAS25, with and without WI, and used the SAI, SICI, and MEP recruitment curves to determine the mechanism underlying priming by WI on PAS25. We demonstrated that SAI, with an inter-stimulus interval of 25 ms, might serve as a predictor of the response to PAS25, whereas I-wave recruitment evaluated by the MEP latency difference was not predictive of the PAS25 response, and found that 15 min WI prior to PAS25 facilitated long-term potentiation (LTP)-like plasticity due to a homeostatic increase in cholinergic activity.

Training intensity-dependent increases in corticospinal but not intracortical excitability after acute strength training.

The purpose of this study was to determine whether the increases in corticospinal excitability (CSE) observed after one session of unilateral isometric strength training (ST) are related to changes in intracortical excitability measured by magnetic brain stimulation (TMS) in the trained and the contralateral untrained biceps brachii (BB) and whether such changes scale with training intensity. On three separate days, 15 healthy young men performed one ST session of 12 sets of eight isometric contractions of the right elbow flexors at 0% (control session), 25%, or 75% of the maximal voluntary contraction (MVC) in a random order. Before and after each session separated at least by 1week, motor evoked potential (MEP) amplitude, short-interval intracortical inhibition (SICI), contralateral silent period (SP), and intracortical facilitation (ICF) generated by TMS were measured in the trained and the untrained BBs. Compared with baseline, MEPs recorded from the trained BB increased by ~47% after training at 75% of MVC (P<.05) but not after training at 0% (~4%) or 25% MVC (~5%, both P>.05). MEPs in the untrained BB and SICI, SP, and ICF in either BB did not change. Therefore, acute high-intensity but not low-intensity unilateral isometric ST increases CSE in the trained BB without modifications in intracortical inhibition or facilitation. Thus, increases in corticospinal neurons or α-α-motoneuron excitability could underlie the increases in CSE. Regardless of contraction intensity, acute isometric ST did not modify the excitability of the ipsilateral primary motor cortex measured by TMS.

Gabapentinoid treatment promotes corticospinal plasticity and regeneration following murine spinal cord injury.

Axon regeneration failure causes neurological deficits and long-term disability after spinal cord injury (SCI). Here, we found that the α2δ2 subunit of voltage-gated calcium channels negatively regulates axon growth and regeneration of corticospinal neurons, the cells that originate the corticospinal tract. Increased α2δ2 expression in corticospinal neurons contributed to loss of corticospinal regrowth ability during postnatal development and after SCI. In contrast, α2δ2 pharmacological blockade through gabapentin administration promoted corticospinal structural plasticity and regeneration in adulthood. Using an optogenetic strategy combined with in vivo electrophysiological recording, we demonstrated that regenerating corticospinal axons functionally integrate into spinal circuits. Mice administered gabapentin recovered upper extremity function after cervical SCI. Importantly, such recovery relies on reorganization of the corticospinal pathway, as chemogenetic silencing of injured corticospinal neurons transiently abrogated recovery. Thus, targeting α2δ2 with a clinically relevant treatment strategy aids repair of motor circuits after SCI.

Motor Cortex Inputs at the Optimum Phase of Beta Cortical Oscillations Undergo More Rapid and Less Variable Corticospinal Propagation.

Brain oscillations involve rhythmic fluctuations of neuronal excitability and may play a crucial role in neural communication. The human corticomuscular system is characterized by beta activity and is readily probed by transcranial magnetic stimulation (TMS). TMS inputs arriving at the excitable phase of beta oscillations in the motor cortex are known to lead to muscle responses of greater amplitude. Here we explore two other possible manifestations of rhythmic excitability in the beta band; windows of reduced response variability and shortened latency. We delivered single-pulse TMS to the motor cortex of healthy human volunteers (10 females and 7 males) during electroencephalography recordings made at rest. TMS delivered at a particular phase of the beta oscillation benefited from not only stronger, but also less variable and more rapid transmission, as evidenced by the greater amplitude, lower coefficient of variation, and shorter latency of motor evoked potentials. Thus, inputs aligned to the optimal phase of the beta EEG in the motor cortex enjoy transmission amplitude gain, but may also benefit from less variability and shortened latencies at subsequent synapses. Neuronal phase may therefore impact corticospinal communication.SIGNIFICANCE STATEMENT Brain oscillations involve rhythmic fluctuations of neuronal excitability. Therefore, motor responses to transcranial magnetic stimulation are larger when a cortical input arrives at a particular phase of the beta activity in the motor cortex. Here, we demonstrate that inputs to corticospinal neurons which coincide with windows of higher excitability also benefit from more rapid and less variable corticospinal transmission. This shortening of latency and increased reproducibility may confer additional advantage to inputs at specific phases. Moreover, these benefits are conserved despite appreciable corticospinal conduction delays.

Mitoautophagy: A Unique Self-Destructive Path Mitochondria of Upper Motor Neurons With TDP-43 Pathology Take, Very Early in ALS.

Mitochondrial dysfunction is one of the converging paths for many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), and TDP-43 pathology is the most common proteinopathy detected in ALS and ALS/Frontotemporal lobar degeneration (ALS/FTLD). We recently identified mitochondrial problems in corticospinal motor neurons (CSMN) and in Betz cells of patients with TDP-43 pathology. However, the timing and the extent of mitochondrial defects, and their mode of degeneration have not been revealed. Because it is important to reveal when problems first begin to emerge and whether they are shared or unique, we investigated the health and integrity of mitochondria in CSMN of prpTDP-43A315T, PFN1G118V, and hSOD1G93A mice at P15 (post-natal day 15)—a very early age in mice without any sign of cellular degeneration.Utilization of immuno-coupled electron microscopy for a detailed surveillance of mitochondria in CSMN and other non-CSMN cells revealed presence of a novel self-destructive path of mitochondrial degeneration, which we named mitoautophagy. Mitoauthopgy is different from mitophagy, as it does not require autophagosome-mediated degradation. In contrast, in this novel path, mitochondria can clear themselves independently. We find that even at this early age, all diseased CSMN begin to display mitochondrial defects, whereas mitochondria in non-CSMN cells are healthy. Our findings not only reveal mitoautophagy as a novel path of mitochondrial clearance that occurs prior to neuronal vulnerability, but it also highlights that it is present mainly in the upper motor neurons of prpTDP-43A315T and PFN1G118V mice, which mimic many aspects of the disease in patients with TDP-43 pathology.

The Recognition of the Distribution Features of Corticospinal Neurons by a Retrograde Trans-synaptic Tracing to Elucidate the Clinical Application of Contralateral Middle Trunk Transfer

Corticospinal neurons (CSNs) undertake direct cortical outputs to the spinal cord and innervate the upper limb through the brachial plexus. Our previous study has shown that the contralateral middle trunk transfer to the paralyzed upper extremity due to cerebral injury can reconstruct the functional cerebral cortex and improve the function of the paralyzed upper extremity. To interpret the cortical reconstruction and the motor improvement after the middle trunk transfer, we explored the distribution of CSNs connecting to the middle, upper, and lower trunk of the brachial plexus by retrograde trans-neuronal tracing using pseudorabies virus (PRV-EGFP or PRV-mRFP). We show that, rather than an individual specific area, these CSNs labelled by each trunk of the brachial plexus were widespread and mainly assembled within the primary motor cortex (M1), secondary motor cortex (M2), primary somatosensory cortex (S1), and slightly within the secondary somatosensory cortex (S2). The three trunk-labelled CSNs were intermingled in these cortices, and mostly connected to more than two trunks, especially the middle trunk-labelled CSNs with higher proportion of co-labelled neurons. Our findings revealed the distribution features of CSNs connecting to the adjacent spinal nerves that innervate the upper limb, which can improve our understanding of the corticospinal circuits associated with motor improvement and the functional cortical reconstruction after the middle trunk transfer.

MCP1-CCR2 and neuroinflammation in the ALS motor cortex with TDP-43 pathology

BackgroundThe involvement of non-neuronal cells and the cells of innate immunity has been attributed to the initiation and progression of ALS. TDP-43 pathology is observed in a broad spectrum of ALS cases and is one of the most commonly shared pathologies. The potential involvement of the neuroimmune axis in the motor cortex of ALS patients with TDP-43 pathology needs to be revealed. This information is vital for building effective treatment strategies.MethodsWe investigated the presence of astrogliosis and microgliosis in the motor cortex of ALS patients with TDP-43 pathology. prpTDP-43A315T-UeGFP mice, corticospinal motor neuron (CSMN) reporter line with TDP-43 pathology, are utilized to reveal the timing and extent of neuroimmune interactions and the involvement of non-neuronal cells to neurodegeneration. Electron microscopy and immunolabeling techniques are used to mark and monitor cells of interest.ResultsWe detected both activated astrocytes and microglia, especially rod-like microglia, in the motor cortex of patients and TDP-43 mouse model. Besides, CCR2+ TMEM119- infiltrating monocytes were detected as they penetrate the brain parenchyma. Interestingly, Betz cells, which normally do not express MCP1, were marked with high levels of MCP1 expression when diseased.ConclusionsThere is an early contribution of a neuroinflammatory response for upper motor neuron (UMN) degeneration with respect to TDP-43 pathology, and MCP1-CCR2 signaling is important for the recognition of diseased upper motor neurons by infiltrating monocytes. The findings are conserved among species and are observed in both ALS and ALS-FTLD patients.

Possible role of backpropagating action potentials in corticospinal neurons in I-wave periodicity following a TMS pulse

A single pulse of TMS or direct electric stimulation over M1 causes repetitive synchronized firing of corticospinal tract (CST) neurons. Two mechanisms for the repetitive firing have been proposed: a cascade of synaptic inputs to the pyramidal neurons and a single reverberating circuit of interneurons. Here, we propose another possibility in which bursting of CST neurons is produced by dendritic Ca2+-spikes. Backpropagation of the initial action potential (I1-wave) from the soma interacts with synaptic input in the dendrites to initiate a dendritic calcium spike. These Ca2+-spikes produce a burst of somatic action potentials that starts about 1.5 ms after the initial discharge of the neuron, which may produce the later I-waves.

Mouse corticospinal system comprises different functional neuronal ensembles depending on their hodology

BackgroundMovement performance depends on the synaptic interactions generated by coherent parallel sensorimotor cortical outputs to different downstream targets. The major outputs of the neocortex to subcortical structures are driven by pyramidal tract neurons (PTNs) located in layer 5B. One of the main targets of PTNs is the spinal cord through the corticospinal (CS) system, which is formed by a complex collection of distinct CS circuits. However, little is known about intracortical synaptic interactions that originate CS commands and how different populations of CS neurons are functionally organized. To further understand the functional organization of the CS system, we analyzed the activity of unambiguously identified CS neurons projecting to different zones of the same spinal cord segment using two-photon calcium imaging and retrograde neuronal tracers.ResultsSensorimotor cortex slices obtained from transgenic mice expressing GCaMP6 funder the Thy1 promoter were used to analyze the spontaneous calcium transients in layer 5 pyramidal neurons. Distinct subgroups of CS neurons projecting to dorsal horn and ventral areas of the same segment show more synchronous activity between them than with other subgroups.ConclusionsThe results indicate that CS neurons projecting to different spinal cord zones segregated into functional ensembles depending on their hodology, suggesting that a modular organization of CS outputs controls sensorimotor behaviors in a coordinated manner.

Skilled Movements in Mice Require Inhibition of Corticospinal Axon Collateral Formation in the Spinal Cord by Semaphorin Signaling.

Corticospinal (CS) neurons in layer V of the sensorimotor cortex are essential for voluntary motor control. Those neurons project axons to specific segments along the rostro-caudal axis of the spinal cord, and reach their spinal targets by sending collateral branches interstitially along axon bundles. Currently, little is known how CS axon collaterals are formed in the proper spinal cord regions. Here, we show that the semaphorin3A (Sema3A)-neuropilin-1 (Npn-1) signaling pathway is an essential negative regulator of CS axon collateral formation in the spinal cord from mice of either sex. Sema3A is expressed in the ventral spinal cord, whereas CS neurons express Npn-1, suggesting that Sema3A might prevent CS axons from entering the ventral spinal cord. Indeed, the ectopic expression of Sema3A in the spinal cord in vivo inhibits CS axon collateral formation, whereas Sema3A or Npn-1 mutant mice have ectopic CS axon collateral formation within the ventral spinal cord compared with littermate controls. Finally, Npn-1 mutant mice exhibit impaired skilled movements, likely because of aberrantly formed CS connections in the ventral spinal cord. These genetic findings reveal that Sema3A-Npn-1 signaling-mediated inhibition of CS axon collateral formation is critical for proper CS circuit formation and the ability to perform skilled motor behaviors.SIGNIFICANCE STATEMENT CS neurons project axons to the spinal cord to control skilled movements in mammals. Previous studies revealed some of the molecular mechanisms underlying different phases of CS circuit development such as initial axon guidance in the brain, and midline crossing in the brainstem and spinal cord. However, the molecular mechanisms underlying CS axon collateral formation in the spinal gray matter has remained obscure. In this study, using in vivo gain-of- and loss-of-function experiments, we show that Sema3A-Npn-1 signaling functions to inhibit CS axon collateral formation in the ventral spinal cord, allowing for the development of proper skilled movements in mice.

Age-related changes in late synaptic inputs to corticospinal neurons and their functional significance: A paired-pulse TMS study

BackgroundRecent work suggests that the function of intracortical interneurons activated by transcranial magnetic stimulation (TMS) is modified in older adults, with the circuits generating short-interval intracortical facilitation (SICF) at longer intervals appearing to be particularly affected. ObjectiveTo use SICF to quantify age-related changes in the excitability and recruitment of late synaptic inputs to corticospinal neurons, and investigate if changes within these circuits contribute to altered motor performance in older adults. MethodsSICF was recorded with 3 different conditioning intensities in 23 young (23.0 ± 4.2 years) and 21 older (67.1 ± 1.1 years) adults. These measures were performed with conventional (posterior-anterior, PA) and reverse (anterior-posterior, AP) current directions using interstimulus intervals targeting late synaptic inputs to corticospinal neurons (3.5–5.3 ms). ResultsPeak SICF recorded with a PA current (SICFPA) was reduced in older adults (P < 0.0001), and occurred at a longer latency (P < 0.05). Furthermore, there was reduced recruitment of SICFPA in older adults (P < 0.0001), but this did not interact with the age-related shift in SICFPA (P = 0.2). In addition, reduced performance on the Purdue pegboard was predicted by increased SICFPA (P < 0.04) occurring at longer latencies (P < 0.04) in old but not young adults. For SICF recorded with an AP current (SICFAP), facilitation was again reduced at longer latencies in older adults (P < 0.0001), but recruitment was not different between groups (P = 0.7) and was unrelated to motor function. ConclusionThese results suggest that there are age-related changes in late synaptic inputs to corticospinal neurons and that these changes influence fine motor performance.

Comparison of gene expression profile of the spinal cord of sprouting-capable neonatal and sprouting-incapable adult mice

BackgroundThe regenerative ability of severed axons in the central nervous system is limited in mammals. However, after central nervous system injury, neural function is partially recovered by the formation of a compensatory neural circuit. In a mouse pyramidotomy model, axonal sprouting of the intact side of the corticospinal tract is observed in the spinal cord, and the axons make new synapses with the denervated side of propriospinal neurons. Moreover, this sprouting ability is enhanced in neonatal mice compared to that in adult mice. Myelin-associated molecules in the spinal cord or intrinsic factors in corticospinal neurons have been investigated in previous studies, but the factors that determine elevated sprouting ability in neonatal mice are not fully understood. Further, in the early phase after pyramidotomy, glial responses are observed in the spinal cord. To elucidate the basal difference in the spinal cord, we compared gene expression profiles of entire C4–7 cervical cord tissues between neonatal (injured at postnatal day 7) and adult (injured at 8 weeks of age) mice by RNA-sequencing. We also tried to identify discordant gene expression changes that might inhibit axonal sprouting in adult mice at the early phase (3 days) after pyramidotomy.ResultsA comparison of neonatal and adult sham groups revealed remarkable basal differences in the spinal cord, such as active neural circuit formation, cell proliferation, the development of myelination, and an immature immune system in neonatal mice compared to that observed in adult mice. Some inflammation-related genes were selectively expressed in adult mice after pyramidotomy, implying the possibility that these genes might be related to the low sprouting ability in adult mice.ConclusionsThis study provides useful information regarding the basal difference between neonatal and adult spinal cords and the possible differential response after pyramidotomy, both of which are necessary to understand why sprouting ability is increased in neonatal mice compared to that in adult mice.

Breaking down the human motor cortex: the layer-specific measurement of corticospinal neuronal activity.

Breaking down the human motor cortex: the layer-specific measurement of corticospinal neuronal activity.

Refinement of the Primate Corticospinal Pathway During Prenatal Development

Perturbation of the developmental refinement of the corticospinal (CS) pathway leads to motor disorders. While non-primate developmental refinement is well documented, in primates invasive investigations of the developing CS pathway have been confined to neonatal and postnatal stages when refinement is relatively modest. Here, we investigated the developmental changes in the distribution of CS projection neurons in cynomolgus monkey (Macaca fascicularis). Injections of retrograde tracer at cervical levels of the spinal cord at embryonic day (E) 95 and E105 show that: (i) areal distribution of back-labeled neurons is more extensive than in the neonate and dense labeling is found in prefrontal, limbic, temporal, and occipital cortex; (ii) distributions of contralateral and ipsilateral projecting CS neurons are comparable in terms of location and numbers of labeled neurons, in contrast to the adult where the contralateral projection is an order of magnitude higher than the ipsilateral projection. Findings from one largely restricted injection suggest a hitherto unsuspected early innervation of the gray matter. In the fetus there was in addition dense labeling in the central nucleus of the amygdala, the hypothalamus, the subthalamic nucleus, and the adjacent region of the zona incerta, subcortical structures with only minor projections in the adult control.