We determined whether rehabilitation after cortical injury also drives dynamic dendritic and spine changes in functionally distinct subsets of neurons, resulting in functional recovery. Moreover, given known requirements for cholinergic systems in mediating complex forms of cortical plasticity, including skilled motor learning, we hypothesized that cholinergic systems are essential mediators of neuronal structural and functional plasticity associated with motor rehabilitation. Adult rats learned a skilled forelimb grasping task and then, underwent destructive lesions of the caudal forelimb region of the motor cortex, resulting in nearly complete loss of grasping ability. Subsequent intensive rehabilitation significantly enhanced both dendritic architecture and spine number in the adjoining rostral forelimb area compared with that in the lesioned animals that were not rehabilitated. Cholinergic ablation markedly attenuated rehabilitation-induced recovery in both neuronal structure and motor function. Thus, rehabilitation focused on an affected limb robustly drives structural compensation in perilesion cortex, enabling functional recovery.

Behavior is derived from the configuration of synaptic connectivity among functionally diverse neurons. Fine motor behavior is absent at birth in most mammals but gradually emerges during subsequent postnatal corticospinal system maturation; the nature of circuit development and reorganization during this period has been largely unexplored. We investigated connectivity and synaptic signaling among functionally distinct corticospinal populations in Fischer 344 rats from postnatal day 18 through 75 using retrograde tracer injections into specific spinal cord segments associated with distinct aspects of forelimb function. Primary motor cortex slices were prepared enabling simultaneous patch-clamp recordings of up to four labeled corticospinal neurons and testing of 3489 potential synaptic connections. We find that, in immature animals, local connectivity is biased toward corticospinal neurons projecting to the same spinal cord segment; this within-population connectivity significantly decreases through maturation until connection frequency is similar between neurons projecting to the same (within-population) or different (across-population) spinal segments. Concomitantly, postnatal maturation is associated with a significant reduction in synaptic efficacy over time and an increase in intrinsic neuronal excitability, altering how excitation is effectively transmitted across recurrent corticospinal networks. Collectively, the postnatal emergence of fine motor control is associated with a relative broadening of connectivity between functionally diverse cortical motor neurons and changes in synaptic properties that could enable the emergence of smaller independent networks, enabling fine motor movement. These changes in synaptic patterning and physiological function provide a basis for the increased capabilities of the mature versus developing brain.

Recurrent synaptic connections between neighboring neurons are a key feature of mammalian cortex, accounting for the vast majority of cortical inputs. Although computational models indicate that reorganization of recurrent connectivity is a primary driver of experience-dependent cortical tuning, the true biological features of recurrent network plasticity are not well identified. Indeed, whether rewiring of connections between cortical neurons occurs during behavioral training, as is widely predicted, remains unknown. Here, we probe M1 recurrent circuits following motor training in adult male rats and find robust synaptic reorganization among functionally related layer 5 neurons, resulting in a 2.5-fold increase in recurrent connection probability. This reorganization is specific to the neuronal subpopulation most relevant for executing the trained motor skill, and behavioral performance was impaired following targeted molecular inhibition of this subpopulation. In contrast, recurrent connectivity is unaffected among neighboring layer 5 neurons largely unrelated to the trained behavior. Training-related corticospinal cells also express increased excitability following training. These findings establish the presence of selective modifications in recurrent cortical networks in adulthood following training.SIGNIFICANCE STATEMENT Recurrent synaptic connections between neighboring neurons are characteristic of cortical architecture, and modifications to these circuits are thought to underlie in part learning in the adult brain. We now show that there are robust changes in recurrent connections in the rat motor cortex upon training on a novel motor task. Motor training results in a 2.5-fold increase in recurrent connectivity, but only within the neuronal subpopulation most relevant for executing the new motor behavior; recurrent connectivity is unaffected among adjoining neurons that do not execute the trained behavior. These findings demonstrate selective reorganization of recurrent synaptic connections in the adult neocortex following novel motor experience, and illuminate fundamental properties of cortical function and plasticity.

Layer 5 neurons of the neocortex receive direct and relatively strong input from the thalamus. However, the intralaminar distribution of these inputs and their capacity for plasticity in adult animals are largely unknown. In slices of the primary motor cortex (M1), we simultaneously recorded from pairs of corticospinal neurons associated with control of distinct motor outputs: distal forelimb versus proximal forelimb. Activation of ChR2-expressing thalamocortical afferents in M1 before motor learning produced equivalent responses in monosynaptic excitation of neurons controlling the distal and proximal forelimb, suggesting balanced thalamic input at baseline. Following skilled grasp training, however, thalamocortical input shifted to bias activation of corticospinal neurons associated with control of the distal forelimb. This increase was associated with a cell-specific increase in mEPSC amplitude but not presynaptic release probability. These findings demonstrate distinct and highly segregated plasticity of thalamocortical projections during adult learning.

A principal objective of spinal cord injury (SCI) research is the restoration of axonal connectivity to denervated targets. We tested the hypothesis that chemotropic mechanisms would guide regenerating spinal cord axons to appropriate brainstem targets. We subjected rats to cervical level 1 (C1) lesions and combinatorial treatments to elicit axonal bridging into and beyond lesion sites. Lentiviral vectors expressing neurotrophin-3 (NT-3) were then injected into an appropriate brainstem target, the nucleus gracilis, and an inappropriate target, the reticular formation. NT-3 expression in the correct target led to reinnervation of the nucleus gracilis in a dose-related fashion, whereas NT-3 expression in the reticular formation led to mistargeting of regenerating axons. Axons regenerating into the nucleus gracilis formed axodendritic synapses containing rounded vesicles, reflective of pre-injury synaptic architecture. Thus, we report for the first time, to the best of our knowledge, the reinnervation of brainstem targets after SCI and an essential role for chemotropic axon guidance in target selection.

We used viral intersectional tools to map the entire projectome of corticospinal neurons associated with fine distal forelimb control in Fischer 344 rats and rhesus macaques. In rats, we found an extraordinarily diverse set of collateral projections from corticospinal neurons to 23 different brain and spinal regions. Remarkably, the vast weighting of this "motor" projection was to sensory systems in both the brain and spinal cord, confirmed by optogenetic and transsynaptic viral intersectional tools. In contrast, rhesus macaques exhibited far heavier and narrower weighting of corticospinal outputs toward spinal and brainstem motor systems. Thus, corticospinal systems in macaques primarily constitute a final output system for fine motor control, whereas this projection in rats exerts a multi-modal integrative role that accesses far broader CNS regions. Unique structural-functional correlations can be achieved by mapping and quantifying a single neuronal system's total axonal output and its relative weighting across CNS targets.