Chronic Adaptations in the Dorsal Horn Following a Cervical Spinal Cord Injury in Primates.

Spinal cord injury (SCI) is devastating, with limited treatment options and variable outcomes. Most in vivo SCI research has focused on the acute and early post-injury periods, and the promotion of axonal growth, so little is understood about the clinically stable chronic state, axonal growth over time, and what plasticity endures. Here, we followed animals into the chronic phase following SCI, to address this gap. Male macaques received targeted deafferentation, affecting three digits of one hand, and were divided into short (4-6 months) or long-term (11-12 months) groups, based on post-injury survival times. Monkeys were assessed behaviorally, where possible, and all exhibited an initial post-injury deficit in manual dexterity, with gradual functional recovery over 2 months. We previously reported extensive sprouting of somatosensory corticospinal (S1 CST) fibers in the dorsal horn in the first five post-injury months. Here, we show that by 1 year, the S1 CST sprouting is pruned, with the terminal territory resembling control animals. This was reflected in the number of putatively "functional" synapses observed, which increased over the first 4-5 months, and then returned to baseline by 1 year. Microglia density also increased in the affected dorsal horn at 4-6 months and then decreased, but did not return to baseline by 1 year, suggesting refinement continues beyond this time. Overall, there is a long period of reorganization and consolidation of adaptive circuitry in the dorsal horn, extending well beyond the initial behavioral recovery. This provides a potential window to target therapeutic opportunities during the chronic phase.

Small sensory spinal lesions that affect hand function in monkeys greatly alter primary afferent and motor neuron connections in the cord.

Small sensory spinal injuries induce plasticity across the neuraxis, but little is understood about their effect on segmental connections or motor neuron (MN) function. Here, we begin to address this at two levels. First, we compared afferent input distributions from the skin and muscles of the digits with corresponding MN pools to determine their spatial relationship, in both the normal state and 4-6 months after a unilateral dorsal root/dorsal column lesion (DRL/DCL), affecting digits 1-3. Second, we looked at specific changes to MN inputs and membrane properties that likely impact functional recovery. Monkeys received a targeted unilateral DRL/DCL, and 4-6 months later, cholera toxin subunit B (CT-B) was injected bilaterally into either the distal pads of digits 1-3, or related intrinsic hand muscles, to label inputs to the cord, and corresponding MNs. In controls (unlesioned side), cutaneous and proprioceptive afferents from digits 1-3 showed different distribution patterns but similar rostrocaudal spread within the dorsal horn from C1 to T2. In contrast, MNs were distributed across just two segments (C7-8). Following the lesion, sensory inputs were significantly diminished across all 10 segments, though this did not alter MN distributions. Afferent and monoamine inputs, as well as KCC2 cotransporters, were also significantly altered on the cell membrane of CT-B labeled MNs postlesion. In contrast, inhibitory neurotransmission and perineuronal net integrity were not altered at this prechronic timepoint.  Our findings indicate that even a small sensory injury can significantly impact sensory and motor spinal neurons and provide new insight into the complex process of recovery.

A Re-evaluation of Whether Non-monosynaptic Homonymous H Reflex Facilitation Tests Propriospinal Circuits.

The C3-C4 propriospinal system is an important pathway mediating movement in cats; it contributes to movements in primates (including humans), and may have a role in recovery after lesion. Validated clinical tests of this system would find many applications, therefore we sought to test whether non-monosynaptic homonymous facilitation of the forearm flexor H reflex is mediated solely via a C3-C4 propriospinal pathway. In one anesthetized macaque monkey, median nerve stimulation elicited an H reflex in the flexor carpi radialis (FCR). Median nerve conditioning stimuli at sub-threshold intensities facilitated the H reflex, for inter-stimulus intervals up to 30 ms. Successive spinal surgical hemisections were then made. C2 lesion left the homonymous facilitation intact, suggesting mediation by spinal, not supraspinal pathways. Facilitation also remained after a second lesion at C5, indicating a major role for segmental (C7-C8) rather than propriospinal (C3-C4) interneurons. In separate experiments in five healthy human subjects, a threshold tracking approach assessed changes in peripheral axon excitability after conditioning stimulation. This was found to be enhanced up to 20 ms after the conditioning stimulus, and could partly, although not completely, underlie the H reflex facilitation seen. We conclude that homonymous facilitation of the H reflex in FCR can be produced by segmental spinal mechanisms, as well as by a supranormal period of nerve excitability. Unfortunately, this straightforward test cannot therefore be used for selective assessment of propriospinal circuits.

Extensive Cortical Convergence to Primate Reticulospinal Pathways.

Early evolution of the motor cortex included development of connections to brainstem reticulospinal neurons; these projections persist in primates. In this study, we examined the organization of corticoreticular connections in five macaque monkeys (one male) using both intracellular and extracellular recordings from reticular formation neurons, including identified reticulospinal cells. Synaptic responses to stimulation of different parts of primary motor cortex (M1) and supplementary motor area (SMA) bilaterally were assessed. Widespread short latency excitation, compatible with monosynaptic transmission over fast-conducting pathways, was observed, as well as longer latency responses likely reflecting a mixture of slower monosynaptic and oligosynaptic pathways. There was a high degree of convergence: 56% of reticulospinal cells with input from M1 received projections from M1 in both hemispheres; for SMA, the equivalent figure was even higher (70%). Of reticulospinal neurons with input from the cortex, 78% received projections from both M1 and SMA (regardless of hemisphere); 83% of reticulospinal cells with input from M1 received projections from more than one of the tested M1 sites. This convergence at the single cell level allows reticulospinal neurons to integrate information from across the motor areas of the cortex, taking account of the bilateral motor context. Reticulospinal connections are known to strengthen following damage to the corticospinal tract, such as after stroke, partially contributing to functional recovery. Extensive corticoreticular convergence provides redundancy of control, which may allow the cortex to continue to exploit this descending pathway even after damage to one area.SIGNIFICANCE STATEMENT The reticulospinal tract (RST) provides a parallel pathway for motor control in primates, alongside the more sophisticated corticospinal system. We found extensive convergent inputs to primate reticulospinal cells from primary and supplementary motor cortex bilaterally. These redundant connections could maintain transmission of voluntary commands to the spinal cord after damage (e.g., after stroke or spinal cord injury), possibly assisting recovery of function.

Reorganization of the Primate Dorsal Horn in Response to a Deafferentation Lesion Affecting Hand Function.

The loss of sensory input following a spinal deafferentation injury can be debilitating, and this is especially true in primates when the hand is involved. Although significant recovery of function occurs, little is currently understood about the reorganization of the neuronal circuitry, particularly within the dorsal horn. This region receives primary afferent input from the periphery, and cortical input via the somatosensory subcomponent of the corticospinal tract (S1 CST), and is critically important in modulating sensory transmission, both in normal and lesioned states. To determine how dorsal horn circuitry alters to facilitate recovery post-injury, we used an established deafferentation lesion model (dorsal root/dorsal column) in male monkeys to remove sensory input from just the opposing digits (digits 1-3) of one hand. This results in a deficit in fine dexterity that recovers over several months. Electrophysiological mapping, tract tracing, and immunolabeling techniques were combined to delineate specific changes to dorsal horn input circuitry. Our main findings show that (1) there is complementary sprouting of the primary afferent and S1 CST populations into an overlapping region of the reorganizing dorsal horn; (2) S1 CST and primary afferent inputs connect in different ways within this region to facilitate sensory integration; and (3) there is a loss of larger S1 CST terminal boutons in the affected dorsal horn, but no change in the size profile of the spared/sprouted primary afferent terminal boutons post-lesion. Understanding such changes helps to inform new and targeted therapies that best promote recovery.SIGNIFICANCE STATEMENT Spinal injuries that remove sensation from the hand, can be debilitating, though functional recovery does occur. We examined changes to the neuronal circuitry of the dorsal horn in monkeys following a lesion that deafferented three digits of one hand. Little is understood about dorsal horn circuitry, despite the fact that this region loses most of its normal input after such an injury, and is clearly a major focus of reorganization. We found that both the spared primary afferents and somatosensory corticospinal efferents sprouted in an overlapping region of the dorsal horn after injury, and that larger (presumably faster) corticospinal terminals are lost, suggesting a significantly altered cortical modulation of primary afferents. Understanding this changing circuitry is important for designing targeted therapies.

Somatosensory corticospinal tract axons sprout within the cervical cord following a dorsal root/dorsal column spinal injury in the rat.

The corticospinal tract (CST) is the major descending pathway controlling voluntary hand function in primates, and though less dominant, it mediates voluntary paw movements in rats. As with primates, the CST in rats originates from multiple (albeit fewer) cortical sites, and functionally different motor and somatosensory subcomponents terminate in different regions of the spinal gray matter. We recently reported in monkeys that following a combined cervical dorsal root/dorsal column lesion (DRL/DCL), both motor and S1 CSTs sprout well beyond their normal terminal range. The S1 CST sprouting response is particularly dramatic, indicating an important, if poorly understood, somatosensory role in the recovery process. As rats are used extensively to model spinal cord injury, we asked if the S1 CST response is conserved in rodents. Rats were divided into sham controls, and two groups surviving post-lesion for ~6 and 10 weeks. A DRL/DCL was made to partially deafferent one paw. Behavioral testing showed a post-lesion deficit and recovery over several weeks. Three weeks prior to ending the experiment, S1 cortex was mapped electrophysiologically, for tracer injection placement to determine S1 CST termination patterns within the cord. Synaptogenesis was also assessed for labeled S1 CST terminals within the dorsal horn. Our findings show that the affected S1 CST sprouts well beyond its normal range in response to a DRL/DCL, much as it does in macaque monkeys. This, along with evidence for increased synaptogenesis post-lesion, indicates that CST terminal sprouting following a central sensory lesion, is a robust and conserved response.

Extensive somatosensory and motor corticospinal sprouting occurs following a central dorsal column lesion in monkeys.

The corticospinal tract (CST) forms the major descending pathway mediating voluntary hand movements in primates, and originates from ∼nine cortical subdivisions in the macaque. While the terminals of spared motor CST axons are known to sprout locally within the cord in response to spinal injury, little is known about the response of the other CST subcomponents. We previously reported that following a cervical dorsal root lesion (DRL), the primary somatosensory (S1) CST terminal projection retracts to 60% of its original terminal domain, while the primary motor (M1) projection remains robust (Darian-Smith et al., J. Neurosci., 2013). In contrast, when a dorsal column lesion (DCL) is added to the DRL, the S1 CST, in addition to the M1 CST, extends its terminal projections bilaterally and caudally, well beyond normal range (Darian-Smith et al., J. Neurosci., 2014). Are these dramatic responses linked entirely to the inclusion of a CNS injury (i.e., DCL), or do the two components summate or interact? We addressed this directly, by comparing data from monkeys that received a unilateral DCL alone, with those that received either a DRL or a combined DRL/DCL. Approximately 4 months post-lesion, the S1 hand region was mapped electrophysiologically, and anterograde tracers were injected bilaterally into the region deprived of normal input, to assess spinal terminal labeling. Using multifactorial analyses, we show that following a DCL alone (i.e., cuneate fasciculus lesion), the S1 and M1 CSTs also sprout significantly and bilaterally beyond normal range, with a termination pattern suggesting some interaction between the peripheral and central lesions.

Classification of Neurons in the Primate Reticular Formation and Changes after Recovery from Pyramidal Tract Lesion.

The reticular formation is important in primate motor control, both in health and during recovery after brain damage. Little is known about the different neurons present in the reticular nuclei. Here we recorded extracellular spikes from the reticular formation in five healthy female awake behaving monkeys (193 cells), and in two female monkeys 1 year after recovery from a unilateral pyramidal tract lesion (125 cells). Analysis of spike shape and four measures derived from the interspike interval distribution identified four clusters of neurons in control animals. Cluster 1 cells had a slow firing rate. Cluster 2 cells had narrow spikes and irregular firing, which often included high-frequency bursts. Cluster 3 cells were highly rhythmic and fast firing. Cluster 4 cells showed negative spikes. A separate population of 42 cells was antidromically identified as reticulospinal neurons in five anesthetized female monkeys. The distribution of spike width in these cells closely overlaid the distribution for cluster 2, leading us tentatively to suggest that cluster 2 included neurons with reticulospinal projections. In animals after corticospinal lesion, cells could be identified in all four clusters. The firing rate of cells in clusters 1 and 2 was increased in lesioned animals relative to control animals (by 52% and 60%, respectively); cells in cluster 2 were also more regular and more bursting in the lesioned animals. We suggest that changes in both membrane properties and local circuits within the reticular formation occur following lesioning, potentially increasing reticulospinal output to help compensate for lost corticospinal descending drive.SIGNIFICANCE STATEMENT This work is the first to subclassify neurons in the reticular formation, providing insights into the local circuitry of this important but little understood structure. The approach developed can be applied to any extracellular recording from this region, allowing future studies to place their data within our current framework of four neural types. Changes in reticular neurons may be important to subserve functional recovery after damage in human patients, such as after stroke or spinal cord injury.

Corticospinal Inputs to Primate Motoneurons Innervating the Forelimb from Two Divisions of Primary Motor Cortex and Area 3a.

Previous anatomical work in primates has suggested that only corticospinal axons originating in caudal primary motor cortex ("new M1") and area 3a make monosynaptic cortico-motoneuronal connections with limb motoneurons. By contrast, the more rostral "old M1" is proposed to control motoneurons disynaptically via spinal interneurons. In six macaque monkeys, we examined the effects from focal stimulation within old and new M1 and area 3a on 135 antidromically identified motoneurons projecting to the upper limb. EPSPs with segmental latency shorter than 1.2 ms were classified as definitively monosynaptic; these were seen only after stimulation within new M1 or at the new M1/3a border (incidence 6.6% and 1.3%, respectively; total n = 27). However, most responses had longer latencies. Using measures of the response facilitation after a second stimulus compared with the first, and the reduction in response latency after a third stimulus compared with the first, we classified these late responses as likely mediated by either long-latency monosynaptic (n = 108) or non-monosynaptic linkages (n = 108). Both old and new M1 generated putative long-latency monosynaptic and non-monosynaptic effects; the majority of responses from area 3a were non-monosynaptic. Both types of responses from new M1 had significantly greater amplitude than those from old M1. We suggest that slowly conducting corticospinal fibers from old M1 generate weak late monosynaptic effects in motoneurons. These may represent a stage in control of primate motoneurons by the cortex intermediate between disynaptic output via an interposed interneuron seen in nonprimates and the fast direct monosynaptic connections present in new M1. SIGNIFICANCE STATEMENT: The corticospinal tract in Old World primates makes monosynaptic connections to motoneurons; previous anatomical work suggests that these connections come only from corticospinal tract (CST) neurons in the subdivision of primary motor cortex within the central sulcus ("new M1") and area 3a. Here, we show using electrophysiology that cortico-motoneuronal connections from fast conducting CST fibers are indeed made exclusively from new M1 and its border with 3a. However, we also show that all parts of M1 and 3a have cortico-motoneuronal connections over more slowly conducting CST axons, as well as exert disynaptic effects on motoneurons via interposed interneurons. Differences between old and new M1 are thus more subtle than previously thought.

Pathways mediating functional recovery.

Following damage to the motor system (e.g., after stroke or spinal cord injury), recovery of upper limb function exploits the multiple pathways which allow motor commands to be sent to the spinal cord. Corticospinal fibers originate from premotor as well as primary motor cortex. While some corticospinal fibers make direct monosynaptic connections to motoneurons, there are also many connections to interneurons which allow control of motoneurons indirectly. Such interneurons may be placed within the cervical enlargement, or more rostrally (propriospinal interneurons). In addition, connections from cortex to the reticular formation in the brainstem allow motor commands to be sent over the reticulospinal tract to these spinal centers. In this review, we consider the relative roles of these different routes for the control of hand function, both in healthy primates and after recovery from lesion.

Blocking central pathways in the primate motor system using high-frequency sinusoidal current.

Electrical stimulation with high-frequency (2-10 kHz) sinusoidal currents has previously been shown to produce a transient and complete nerve block in the peripheral nervous system. Modeling and in vitro studies suggest that this is due to a prolonged local depolarization across a broad section of membrane underlying the blocking electrode. Previous work has used cuff electrodes wrapped around the peripheral nerve to deliver the blocking stimulus. We extended this technique to central motor pathways, using a single metal microelectrode to deliver focal sinusoidal currents to the corticospinal tract at the cervical spinal cord in anesthetized adult baboons. The extent of conduction block was assessed by stimulating a second electrode caudal to the blocking site and recording the antidromic field potential over contralateral primary motor cortex. The maximal block achieved was 99.6%, similar to findings of previous work in peripheral fibers, and the optimal frequency for blocking was 2 kHz. Block had a rapid onset, being complete as soon as the transient activation associated with the start of the sinusoidal current was over. High-frequency block was also successfully applied to the pyramidal tract at the medulla, ascending sensory pathways in the dorsal columns, and the descending systems of the medial longitudinal fasciculus. High-frequency sinusoidal stimulation produces transient, reversible lesions in specific target locations and therefore could be a useful alternative to permanent tissue transection in some experimental paradigms. It also could help to control or prevent some of the hyperactivity associated with chronic neurological disorders.

Differences between Han Chinese and Caucasians in transcranial magnetic stimulation parameters.

The study was conducted to investigate the difference between Han Chinese and Caucasians on various parameters measured from responses to transcranial magnetic brain stimulation (TMS). Sixteen subjects were studied in each group. A circular coil at the vertex was used for stimulation, whilst recording surface electromyograms from right first dorsal interosseous. In the passive state, motor-evoked potential (MEP) threshold, MEP recruitment, short-interval intracortical inhibition (SICI) and intracortical facilitation were measured. The MEP threshold, recruitment and silent period were also measured in the active state. Chinese subjects showed significantly higher passive thresholds (P < 0.005), less inhibition of the motor response (SICI, P < 0.0005) and a shorter silent period (P < 0.05). Differences in SICI appeared to be a consequence of the differences in passive threshold and were not seen when active threshold was used to determine the conditioning stimulus intensity. Differences in silent period may also reflect differences in cortical excitability rather than inhibitory processes, as they were not seen when the silent-period duration was expressed as a function of MEP size, rather than TMS intensity. There appears to be a significant difference in some TMS parameters between Han Chinese and Caucasian subjects. This may reflect an underlying difference in cortical excitability.

Reticular formation responses to magnetic brain stimulation of primary motor cortex.

Transcranial magnetic stimulation (TMS) of cerebral cortex is a popular technique for the non-invasive investigation of motor function. TMS is often assumed to influence spinal circuits solely via the corticospinal tract. We were interested in possible trans-synaptic effects of cortical TMS on the ponto-medullary reticular formation in the brainstem, which is the source of the reticulospinal tract and could also generate spinal motor output. We recorded from 210 single units in the reticular formation of three anaesthetized macaque monkeys whilst TMS was performed over primary motor cortex. Short latency responses were observed consistent with activation of a cortico-reticular pathway. However, we also demonstrated surprisingly powerful responses at longer latency, which often appeared at lower threshold than the earlier effects. These late responses seemed to be generated partly as a consequence of the sound click made by coil discharge, and changed little with coil location. This novel finding has implications for the design of future studies using TMS, as well as suggesting a means of non-invasively probing an otherwise inaccessible important motor centre.

Beta-band intermuscular coherence: a novel biomarker of upper motor neuron dysfunction in motor neuron disease.

In motor neuron disease, the focus of therapy is to prevent or slow neuronal degeneration with neuroprotective pharmacological agents; early diagnosis and treatment are thus essential. Incorporation of needle electromyographic evidence of lower motor neuron degeneration into diagnostic criteria has undoubtedly advanced diagnosis, but even earlier diagnosis might be possible by including tests of subclinical upper motor neuron disease. We hypothesized that beta-band (15-30 Hz) intermuscular coherence could be used as an electrophysiological marker of upper motor neuron integrity in such patients. We measured intermuscular coherence in eight patients who conformed to established diagnostic criteria for primary lateral sclerosis and six patients with progressive muscular atrophy, together with 16 age-matched controls. In the primary lateral sclerosis variant of motor neuron disease, there is selective destruction of motor cortical layer V pyramidal neurons and degeneration of the corticospinal tract, without involvement of anterior horn cells. In progressive muscular atrophy, there is selective degeneration of anterior horn cells but a normal corticospinal tract. All patients with primary lateral sclerosis had abnormal motor-evoked potentials as assessed using transcranial magnetic stimulation, whereas these were similar to controls in progressive muscular atrophy. Upper and lower limb intermuscular coherence was measured during a precision grip and an ankle dorsiflexion task, respectively. Significant beta-band coherence was observed in all control subjects and all patients with progressive muscular atrophy tested, but not in the patients with primary lateral sclerosis. We conclude that intermuscular coherence in the 15-30 Hz range is dependent on an intact corticospinal tract but persists in the face of selective anterior horn cell destruction. Based on the distributions of coherence values measured from patients with primary lateral sclerosis and control subjects, we estimated the likelihood that a given measurement reflects corticospinal tract degeneration. Therefore, intermuscular coherence has potential as a quantitative test of subclinical upper motor neuron involvement in motor neuron disease.

Corticospinal activation confounds cerebellar effects of posterior fossa stimuli.

OBJECTIVE: To investigate the efficacy of magnetic stimulation over the posterior fossa (PF) as a non-invasive assessment of cerebellar function in man. METHODS: We replicated a previously reported conditioning-test paradigm in 11 healthy subjects. Transcranial magnetic stimulation (TMS) at varying intensities was applied to the PF and motor cortex with a 3, 5 or 7 ms interstimulus interval (ISI), chosen randomly for each trial. Surface electromyogram (EMG) activity was recorded from two intrinsic hand muscles and two forearm muscles. Responses were averaged and rectified, and MEP amplitudes were compared to assess whether suppression of the motor output occurred as a result of the PF conditioning pulse. RESULTS: Cortical MEPs were suppressed following conditioning-test ISIs of 5 or 7 ms. No suppression occurred with an ISI of 3 ms. PF stimuli alone also produced EMG responses, suggesting direct activation of the corticospinal tract (CST). CONCLUSIONS: CST collaterals are known to contact cortical inhibitory interneurones; antidromic CST activation could therefore contribute to the observed suppression of cortical MEPs. SIGNIFICANCE: PF stimulation probably activates multiple pathways; even at low intensities it should not be regarded as a selective assessment of cerebellar function unless stringent controls can confirm the absence of confounding activity in other pathways.