Basal Forebrain Cholinergic Neurons Selectively Drive Coordinated Motor Learning in Mice.

Motor control requires precise temporal and spatial encoding across distinct motor centers that is refined through the repetition of learning. The recruitment of motor regions requires modulatory input to shape circuit activity. Here, we identify a role for the baso-cortical cholinergic pathway in the acquisition of a coordinated motor skill in mice. Targeted depletion of basal forebrain cholinergic neurons results in significant impairments in training on the rotarod task of coordinated movement. Cholinergic neuromodulation is required during training sessions as chemogenetic inactivation of cholinergic neurons also impairs task acquisition. Rotarod learning is known to drive refinement of corticostriatal neurons arising in both medial prefrontal cortex (mPFC) and motor cortex, and we have found that cholinergic input to both motor regions is required for task acquisition. Critically, the effects of cholinergic neuromodulation are restricted to the acquisition stage, as depletion of basal forebrain cholinergic neurons after learning does not affect task execution. Our results indicate a critical role for cholinergic neuromodulation of distant cortical motor centers during coordinated motor learning.SIGNIFICANCE STATEMENT Acetylcholine release from basal forebrain cholinergic neuron terminals rapidly modulates neuronal excitability, circuit dynamics, and cortical coding; all processes required for processing complex sensory information, cognition, and attention. We found that depletion or transient silencing of cholinergic inputs to anatomically isolated motor areas, medial prefrontal cortex (mPFC) and motor cortex, selectively led to significant impairments on coordinated motor learning; disrupting this baso-cortical network after acquisition elicited no effect on task execution. Our results indicate a pivotal role for cholinergic neuromodulation of distant cortical motor centers during coordinated motor learning. These findings support the concept that cognitive components (such as attention) are indispensable in the adjustment of motor output and training-induced improvements in motor performance.

Reinduced Wnt signaling limits regenerative potential of sensory axons in the spinal cord following conditioning lesion.

Conditioning lesion of the peripheral branch of dorsal column axons is a well-known paradigm enabling the central branch to regenerate after injury to the spinal cord. However, only a small number of regenerating axons enter grafted substrates, and they do not grow beyond the lesion. We found that conditioning lesion induces, in addition to growth-stimulating genes, related to receptor tyrosine kinase (Ryk), a potent repulsive receptor for Wnts. Wnts are expressed around the site of spinal cord injury, and we found that grafted bone marrow stromal cells secreting the Wnt inhibitors secreted frizzled-related protein 2 or Wnt inhibitory factor 1 enhanced regeneration of the central branch after peripheral conditioning lesion. Furthermore, we found that Wnt4-expressing grafts caused dramatic long-range retraction of the injured central branch of conditioned dorsal root ganglion neurons. Macrophages accumulate along the path of receding axons but not around Wnt4-expressing cells, suggesting that the retraction of dorsal column axons is not a secondary effect of increased macrophages attracted by Wnt4. Therefore, Wnt-Ryk signaling is an inhibitory force co-induced with growth-stimulating factors after conditioning lesion. Overcoming Wnt inhibition may further enhance therapies being designed on the basis of the conditioning-lesion paradigm.

Corticospinal Modulation of Precision Movements.

Recently we demonstrated a critical role for temporal coding of corticospinal activity in a prehension movement requiring precise forelimb control. Learning of precision isometric pull drives large-scale remodeling of corticospinal motor networks. Optogenetic modulation of corticospinal activity and full transection of the corticospinal tract disrupted critical functions of the network in expert animals resulting in impaired modulation of precise movements. In contrast, we observed more widespread corticospinal co-activation and limited temporal coding on a similar, yet more simplistic prehension task, adaptive isometric pull. Disrupting corticospinal neuron activity had much more limited effects on adaptive isometric pull, which was found to be corticospinal independent by transection of the corticospinal tract. Here we discuss these results in context of known roles for corticospinal and corticostriatal neurons in motor control, as well as some of the questions our study raised.

A Kinematic Deviation Index (KDI) for Evaluation of Forelimb Function in Rodents.

Rodent models are widely used to study neurological conditions and assess forelimb movement to measure function performance, deficit, recovery and treatment effectiveness. Traditional assessment methods based on endpoints such as whether the task is accomplished, while easy to implement, provide limited information on movement patterns important to assess different functional strategies. On the other side, detailed kinematic analysis provides granular information on the movement patterns but is difficult to compare across laboratories, and may not translate to clinical metrics of upper limb function. To address these limitations, we developed and validated a kinematic deviation index (KDI) for rodents that mimics current trends in clinical research. The KDI is a unitless summary score that quantifies the difference between an animal movement during a task and its optimal performance derived from spatiotemporal marker sequences without pre-specifying movements. We demonstrate the utility of KDI in assessing reaching and grasping in mice and validate its discrimination between trial endpoints in healthy animals. Furthermore, we show KDI sensitivity to interventions, including acute and chronic spinal cord injury and optogenetic disruption of sensorimotor circuits. The KDI provides a comprehensive measure of motor function that bridges the gap between detailed kinematic analysis and simple success/failure metrics, offering a valuable tool for assessing recovery and compensation in rodent models of neurological disorders.

Task-specific modulation of corticospinal neuron activity during motor learning in mice.

Motor skill learning relies on the plasticity of the primary motor cortex as task acquisition drives cortical motor network remodeling. Large-scale cortical remodeling of evoked motor outputs occurs during the learning of corticospinal-dependent prehension behavior, but not simple, non-dexterous tasks. Here we determine the response of corticospinal neurons to two distinct motor training paradigms and assess the role of corticospinal neurons in the execution of a task requiring precise modulation of forelimb movement and one that does not. In vivo calcium imaging in mice revealed temporal coding of corticospinal activity coincident with the development of precise prehension movements, but not more simplistic movement patterns. Transection of the corticospinal tract and optogenetic regulation of corticospinal activity show the necessity for patterned corticospinal network activity in the execution of precise movements but not simplistic ones. Our findings reveal a critical role for corticospinal network modulation in the learning and execution of precise motor movements.

Induction of corticospinal regeneration by lentiviral trkB-induced Erk activation.

Several experimental manipulations of the CNS environment successfully elicit regeneration of sensory and bulbospinal motor axons but fail to elicit regeneration of corticospinal axons, suggesting that cell-intrinsic mechanisms limit the regeneration of this critical class of motor neurons. We hypothesized that enhancement of intrinsic neuronal growth mechanisms would enable adult corticospinal motor axon regeneration. Lentiviral vectors were used to overexpress the BDNF receptor trkB in layer V corticospinal motor neurons. After subcortical axotomy, trkB transduction induced corticospinal axon regeneration into subcortical lesion sites expressing BDNF. In the absence of trkB overexpression, no regeneration occurred. Selective deletion of canonical, trkB-mediated neurite outgrowth signaling by mutation of the Shc/FRS-2 activation domain prohibited Erk activation and eliminated regeneration. These findings support the hypothesis that the refractory regenerative state of adult corticospinal axons can be attributed at least in part to neuron-intrinsic mechanisms, and that activation of ERK signaling can elicit corticospinal tract regeneration.

Transient demyelination increases the efficiency of retrograde AAV transduction.

Adeno-associated virus (AAV) is capable of mediating retrograde viral transduction of central and peripheral neurons. This occurs at a relatively low efficiency, which we previously found to be dependent upon capsid serotype. We sought to augment retrograde transduction by providing increased axonal access to peripherally delivered AAV. Others have described utilizing full transection of peripheral nerves to mediate retrograde viral transduction of motor neurons. Here, we examined the ability of a transient demyelinating event to modulate levels of retrograde AAV transduction. Transient demyelination does not cause lasting functional deficits. Ethidium bromide (EtBr)-induced transient demyelination of the sciatic nerve resulted in significant elevation of retrograde transduction of both motor and sensory neurons. Retrograde transduction levels of motor neurons and heavily myelinated, large-diameter sensory neurons increased at least sixfold following peripheral delivery of self-complementary AAV serotype 1 (scAAV1) and serotype 2 (scAAV2), when preceded by demyelination. These findings identify a means of significantly enhancing retrograde vector transport for use in experimental paradigms requiring either retrograde neuronal identification and gene expression, or translational treatment paradigms.

Sensory Circuit Remodeling and Movement Recovery After Spinal Cord Injury.

Restoring sensory circuit function after spinal cord injury (SCI) is essential for recovery of movement, yet current interventions predominantly target motor pathways. Integrated cortical sensorimotor networks, disrupted by SCI, are critical for perceiving, shaping, and executing movement. Corticocortical connections between primary sensory (S1) and motor (M1) cortices are critical loci of functional plasticity in response to learning and injury. Following SCI, in the motor cortex, corticocortical circuits undergo dynamic remodeling; however, it remains unknown how rehabilitation shapes the plasticity of S1-M1 networks or how these changes may impact recovery of movement.

Functional Electrical Stimulation and the Modulation of the Axon Regeneration Program.

Neural injury in mammals often leads to persistent functional deficits as spontaneous repair in the peripheral nervous system (PNS) is often incomplete, while endogenous repair mechanisms in the central nervous system (CNS) are negligible. Peripheral axotomy elicits growth-associated gene programs in sensory and motor neurons that can support reinnervation of peripheral targets given sufficient levels of debris clearance and proximity to nerve targets. In contrast, while damaged CNS circuitry can undergo a limited amount of sprouting and reorganization, this innate plasticity does not re-establish the original connectivity. The utility of novel CNS circuitry will depend on effective connectivity and appropriate training to strengthen these circuits. One method of enhancing novel circuit connectivity is through the use of electrical stimulation, which supports axon growth in both central and peripheral neurons. This review will focus on the effects of CNS and PNS electrical stimulation in activating axon growth-associated gene programs and supporting the recovery of motor and sensory circuits. Electrical stimulation-mediated neuroplasticity represents a therapeutically viable approach to support neural repair and recovery. Development of appropriate clinical strategies employing electrical stimulation will depend upon determining the underlying mechanisms of activity-dependent axon regeneration and the heterogeneity of neuronal subtype responses to stimulation.

Analysis of the immune response to sciatic nerve injury identifies efferocytosis as a key mechanism of nerve debridement.

Sciatic nerve crush injury triggers sterile inflammation within the distal nerve and axotomized dorsal root ganglia (DRGs). Granulocytes and pro-inflammatory Ly6Chigh monocytes infiltrate the nerve first and rapidly give way to Ly6Cnegative inflammation-resolving macrophages. In axotomized DRGs, few hematogenous leukocytes are detected and resident macrophages acquire a ramified morphology. Single-cell RNA-sequencing of injured sciatic nerve identifies five macrophage subpopulations, repair Schwann cells, and mesenchymal precursor cells. Macrophages at the nerve crush site are molecularly distinct from macrophages associated with Wallerian degeneration. In the injured nerve, macrophages 'eat' apoptotic leukocytes, a process called efferocytosis, and thereby promote an anti-inflammatory milieu. Myeloid cells in the injured nerve, but not axotomized DRGs, strongly express receptors for the cytokine GM-CSF. In GM-CSF-deficient (Csf2-/-) mice, inflammation resolution is delayed and conditioning-lesion-induced regeneration of DRG neuron central axons is abolished. Thus, carefully orchestrated inflammation resolution in the nerve is required for conditioning-lesion-induced neurorepair.

Efficient retrograde neuronal transduction utilizing self-complementary AAV1.

Adeno-associated virus (AAV) is frequently used for gene transfer into the central nervous system (CNS). Similar to adenovirus and rabies virus, AAV can be taken up by axons and retrogradely transported, resulting in neuronal gene expression distant from the injection site. We investigated the retrograde transport properties of self-complementary AAV (scAAV) serotypes 1-6 following peripheral injection. Injection of scAAV into either rat extensor carpi muscle or sciatic nerve resulted in detectable retrograde vector transport and reporter gene expression in spinal cord motor neurons (MNs). Serotype 1 resulted in the highest level of retrograde transport, with 4.1 +/- 0.3% of cervical MNs projecting to the extensor carpi transduced following intramuscular injection, and 7.5 +/- 3.1% of lumbar MNs transduced after sciatic nerve injection. In contrast to scAAV1, retrograde transduction with scAAV2 was undetectable following intramuscular injection, and was detected in only 0.81 +/- 0.15% of MNs projecting to the sciatic nerve following intranerve injection. Furthermore, sciatic injection of single-stranded AAV1 required injection of tenfold higher numbers of viral particles for detectable transgene expression compared to scAAV1, and then only 0.91 +/- 0.24% of lumbar MNs were transduced. Our data provide the basis for increased retrograde transduction efficiency using peripheral injections of scAAV1 vectors for therapeutic gene delivery to the spinal cord.

Cortical Reorganization of Sensorimotor Systems and the Role of Intracortical Circuits After Spinal Cord Injury.

The plasticity of sensorimotor systems in mammals underlies the capacity for motor learning as well as the ability to relearn following injury. Spinal cord injury, which both deprives afferent input and interrupts efferent output, results in a disruption of cortical somatotopy. While changes in corticospinal axons proximal to the lesion are proposed to support the reorganization of cortical motor maps after spinal cord injury, intracortical horizontal connections are also likely to be critical substrates for rehabilitation-mediated recovery. Intrinsic connections have been shown to dictate the reorganization of cortical maps that occurs in response to skilled motor learning as well as after peripheral injury. Cortical networks incorporate changes in motor and sensory circuits at subcortical or spinal levels to induce map remodeling in the neocortex. This review focuses on the reorganization of cortical networks observed after injury and posits a role of intracortical circuits in recovery.

Corticospinal circuit plasticity in motor rehabilitation from spinal cord injury.

Restoring corticospinal function after spinal cord injury is a significant challenge as the corticospinal tract elicits no substantive, spontaneous regeneration, and its interruption leaves a permanent deficit. The corticospinal circuit serves multiple motor and sensory functions within the mammalian nervous system as the direct link between isocortex and spinal cord. Maturation of the corticospinal circuit involves the refinement of projections within the spinal cord and a subsequent refinement of motor maps within the cortex. The plasticity of these cortical motor maps mirrors the acquisition of skilled motor learning, and both the maps and motor skills are disrupted following injury to the corticospinal tract. The motor cortex exhibits the capacity to incorporate changes in corticospinal projections induced by both spontaneous and therapeutic-mediated plasticity of corticospinal axons through appropriate rehabilitation. An understanding of the mechanisms of corticospinal plasticity in motor learning will undoubtedly help inform strategies to improve motor rehabilitation after spinal cord injury.

Axon Guidance Molecules and Neural Circuit Remodeling After Spinal Cord Injury.

…once the development was ended, the founts of growth and regeneration of the axons and dendrites dried up irrevocably. Santiago Ramón y Cajal Cajal's neurotropic theory postulates that the complexity of the nervous system arises from the collaboration of neurotropic signals from neuronal and non-neuronal cells and that once development has ended, a paucity of neurotropic signals means that the pathways of the central nervous system are "fixed, ended, immutable". While the capacity for regeneration and plasticity of the central nervous system may not be quite as paltry as Cajal proposed, regeneration is severely limited in scope as there is no spontaneous regeneration of long-distance projections in mammals and therefore limited opportunity for functional recovery following spinal cord injury. It is not a far stretch from Cajal to hypothesize that reappropriation of the neurotropic programs of development may be an appropriate strategy for reconstitution of injured circuits. It has become clear, however, that a significant number of the molecular cues governing circuit development become re-active after injury and many assume roles that paradoxically obstruct the functional re-wiring of severed neural connections. Therefore, the problem to address is how individual neural circuits respond to specific molecular cues following injury, and what strategies will be necessary for instigating functional repair or remodeling of the injured spinal cord.

Ryk controls remapping of motor cortex during functional recovery after spinal cord injury.

Limited functional recovery can be achieved through rehabilitation after incomplete spinal cord injury. Eliminating the function of a repulsive Wnt receptor, Ryk, in mice and rats by either conditional knockout in the motor cortex or monoclonal antibody infusion resulted in increased corticospinal axon collateral branches with presynaptic puncta in the spinal cord and enhanced recovery of forelimb reaching and grasping function following a cervical dorsal column lesion. Using optical stimulation, we observed that motor cortical output maps underwent massive changes after injury and that hindlimb cortical areas were recruited to control the forelimb over time. Furthermore, a greater cortical area was dedicated to controlling the forelimb in Ryk conditional knockout mice than in controls (wild-type or heterozygotes). In the absence of weekly task-specific training, recruitment of ectopic cortical areas was greatly reduced and there was no significant functional recovery even in Ryk conditional knockout mice. Our study provides evidence that maximal circuit reorganization and functional recovery can be achieved by combining molecular manipulation and targeted rehabilitation.

A novel and robust conditioning lesion induced by ethidium bromide.

Molecular and cellular mechanisms underlying the peripheral conditioning lesion remain unsolved. We show here that injection of a chemical demyelinating agent, ethidium bromide, into the sciatic nerve induces a similar set of regeneration-associated genes and promotes a 2.7-fold greater extent of sensory axon regeneration in the spinal cord than sciatic nerve crush. We found that more severe peripheral demyelination correlates with more severe functional and electrophysiological deficits, but more robust central regeneration. Ethidium bromide injection does not activate macrophages at the demyelinated sciatic nerve site, as observed after nerve crush, but briefly activates macrophages in the dorsal root ganglion. This study provides a new method for investigating the underlying mechanisms of the conditioning response and suggests that loss of the peripheral myelin may be a major signal to change the intrinsic growth state of adult sensory neurons and promote regeneration.

Remodelling of spared proprioceptive circuit involving a small number of neurons supports functional recovery.

Studies show that limited functional recovery can be achieved by plasticity and adaptation of the remaining circuitry in partial injuries in the central nervous system, although the new circuits that arise in these contexts have not been clearly identified or characterized. We show here that synaptic contacts from dorsal root ganglions to a small number of dorsal column neurons, a caudal extension of nucleus gracilis, whose connections to the thalamus are spared in a precise cervical level 1 lesion, underwent remodeling over time. These connections support proprioceptive functional recovery in a conditioning lesion paradigm, as silencing or eliminating the remodelled circuit completely abolishes the recovered proprioceptive function of the hindlimb. Furthermore, we show that blocking repulsive Wnt signalling increases axon plasticity and synaptic connections that drive greater functional recovery.

Axon guidance and injury-lessons from Wnts and Wnt signaling.

Many studies in the past decade have revealed the role and mechanisms of Wnt signaling in axon guidance during development and the reinduction of Wnt signaling in adult central nervous system axons upon traumatic injury, which has profound influences on axon regeneration. With 19 Wnts and 14 known receptors (10 Frizzleds (Fzds), Ryk, Ror1/2 and PTK7), the Wnt family signaling proteins contribute significantly to the wiring specificity of the complex brain and spinal cord circuitry. Subsequent investigation into the signaling mechanisms showed that conserved cell polarity pathways mediate growth cone steering. These cell polarity pathways may unveil general principles of growth cone guidance. The reappeared Wnt signaling system after spinal cord injury limits the regrowth of both descending and ascending motor and sensory axons. Therefore, the knowledge of Wnt signaling mechanisms learned from axon development can be applied to axon repair in adulthood.

Expression of the Wnt signaling system in central nervous system axon guidance and regeneration.

Wnt signaling is essential for axon wiring throughout the development of the nervous system in vertebrates and invertebrates. In rodents, Wnts are expressed in gradients that span the entire anterior-posterior (A-P) axis in the spinal cord and the medial-lateral axis in the superior colliculus. In the brainstem, Wnts are expressed in more complex gradients along the A-P axis. These gradients provide directional information for axon pathfinding and positional information for topographic mapping and are detected by cell polarity signaling pathways in the growth cone. The gradient expression of Wnts and the coordinated expression of Wnt signaling systems are regulated by mechanisms which are currently unknown. Injury to the adult spinal cord results in the re-induction of Wnts in multiple cell types around the lesion site and their signaling system in injured axons. The re-induced Wnts form gradients around the lesion site, with the lesion site being the peak. The re-inducedWnts may be responsible for the well-known retraction of descending motor axons through the receptor Ryk (related receptor tyrosine kinases). Wnt signaling is an appealing new therapeutic target for CNS repair. The mechanisms regulating the re-induction are unknown but will be informative for therapeutic design.