Long-distance growth and connectivity of neural stem cells after severe spinal cord injury.

Neural stem cells (NSCs) expressing GFP were embedded into fibrin matrices containing growth factor cocktails and grafted to sites of severe spinal cord injury. Grafted cells differentiated into multiple cellular phenotypes, including neurons, which extended large numbers of axons over remarkable distances. Extending axons formed abundant synapses with host cells. Axonal growth was partially dependent on mammalian target of rapamycin (mTOR), but not Nogo signaling. Grafted neurons supported formation of electrophysiological relays across sites of complete spinal transection, resulting in functional recovery. Two human stem cell lines (566RSC and HUES7) embedded in growth-factor-containing fibrin exhibited similar growth, and 566RSC cells supported functional recovery. Thus, properties intrinsic to early-stage neurons can overcome the inhibitory milieu of the injured adult spinal cord to mount remarkable axonal growth, resulting in formation of new relay circuits that significantly improve function. These therapeutic properties extend across stem cell sources and species.

Reorganization of Recurrent Layer 5 Corticospinal Networks Following Adult Motor Training.

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.

Rehabilitation drives enhancement of neuronal structure in functionally relevant neuronal subsets.

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.

Motor cortex maturation is associated with reductions in recurrent connectivity among functional subpopulations and increases in intrinsic excitability.

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.

Cholinergic systems are essential for late-stage maturation and refinement of motor cortical circuits.

Previous studies reported that early postnatal cholinergic lesions severely perturb early cortical development, impairing neuronal cortical migration and the formation of cortical dendrites and synapses. These severe effects of early postnatal cholinergic lesions preclude our ability to understand the contribution of cholinergic systems to the later-stage maturation of topographic cortical representations. To study cholinergic mechanisms contributing to the later maturation of motor cortical circuits, we first characterized the temporal course of cortical motor map development and maturation in rats. In this study, we focused our attention on the maturation of cortical motor representations after postnatal day 25 (PND 25), a time after neuronal migration has been accomplished and cortical volume has reached adult size. We found significant maturation of cortical motor representations after this time, including both an expansion of forelimb representations in motor cortex and a shift from proximal to distal forelimb representations to an extent unexplainable by simple volume enlargement of the neocortex. Specific cholinergic lesions placed at PND 24 impaired enlargement of distal forelimb representations in particular and markedly reduced the ability to learn skilled motor tasks as adults. These results identify a novel and essential role for cholinergic systems in the late refinement and maturation of cortical circuits. Dysfunctions in this system may constitute a mechanism of late-onset neurodevelopmental disorders such as Rett syndrome and schizophrenia.

Structural plasticity within highly specific neuronal populations identifies a unique parcellation of motor learning in the adult brain.

Cortical networks undergo adaptations during learning, including increases in dendritic complexity and spines. We hypothesized that structural elaborations during learning are restricted to discrete subsets of cells preferentially activated by, and relevant to, novel experience. Accordingly, we examined corticospinal motor neurons segregated on the basis of their distinct descending projection patterns, and their contribution to specific aspects of motor control during a forelimb skilled grasping task in adult rats. Learning-mediated structural adaptations, including extensive expansions of spine density and dendritic complexity, were restricted solely to neurons associated with control of distal forelimb musculature required for skilled grasping; neurons associated with control of proximal musculature were unchanged by the experience. We further found that distal forelimb-projecting and proximal forelimb-projecting neurons are intermingled within motor cortex, and that this distribution does not change as a function of skill acquisition. These findings indicate that representations of novel experience in the adult motor cortex are associated with selective structural expansion in networks of functionally related, active neurons that are distributed across a single cortical domain. These results identify a distinct parcellation of cortical resources in support of learning.

Rhesus macaque versus rat divergence in the corticospinal projectome.

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.

Modulation of tactile feedback for the execution of dexterous movement.

Although dexterity relies on the constant transmission of sensory information, unchecked feedback can be disruptive. Yet how somatosensory feedback from the hands is regulated and whether this modulation influences movement remain unclear. We found that mouse tactile afferents recruit neurons in the brainstem cuneate nucleus, whose activity is modulated by distinct classes of local inhibitory neurons. Manipulation of these inhibitory circuits suppresses or enhances the transmission of tactile information, which affects manual behaviors. Top-down cortical pathways innervate cuneate in a complementary pattern, with somatosensory cortical neurons targeting the core tactile region of cuneate and a large rostral cortical population driving feed-forward inhibition of tactile transmission through an inhibitory shell. These findings identify a circuit basis for tactile feedback modulation that enables the effective execution of dexterous movement.

The basal forebrain cholinergic system is required specifically for behaviorally mediated cortical map plasticity.

The basal forebrain cholinergic system has been implicated in the reorganization of adult cortical sensory and motor representations under many, but not all, experimental conditions. It is still not fully understood which types of plasticity require the cholinergic system and which do not. In this study, we examine the hypothesis that the basal forebrain cholinergic system is required for eliciting plasticity associated with complex cognitive processing (e.g., behavioral experiences that drive cortical reorganization) but is not required for plasticity mediated under behaviorally independent conditions. We used established experimental manipulations to elicit two distinct forms of plasticity within the motor cortex: facial nerve transections evoke reorganization of cortical motor representations independent of behavioral experience, and skilled forelimb training induces behaviorally dependent expansion of forelimb motor representations. In animals that underwent skilled forelimb training in conjunction with a facial nerve lesion, cholinergic mechanisms were required for mediating the behaviorally dependent plasticity associated with the skilled motor training but were not necessary for mediating plasticity associated with the facial nerve transection. These results dissociate the contribution of cholinergic mechanisms to distinct forms of cortical plasticity and support the hypothesis that the forebrain cholinergic system is selectively required for modulating complex forms of cortical plasticity driven by behavioral experience.

NGF is essential for hippocampal plasticity and learning.

Nerve growth factor (NGF) is produced in the hippocampus throughout life and is retrogradely trafficked to septal cholinergic neurons, providing a potential mechanism for modulating cholinergic inputs and, thereby, hippocampal plasticity. To explore NGF modulation of hippocampal plasticity and function, NGF levels were augmented or blocked in intact adult rats, and subsequent in vivo effects on cholinergic neurons, hippocampal long-term potentiation (LTP), and learning were examined. NGF augmentation significantly enhanced cholinergic neuronal markers and facilitated induction of hippocampal LTP. Blockade of endogenous NGF significantly reduced hippocampal LTP and impaired retention of spatial memory. These findings reveal an essential role for NGF in regulating biological mechanisms related to plasticity and memory in the intact adult brain.

The basal forebrain cholinergic system is essential for cortical plasticity and functional recovery following brain injury.

A reorganization of cortical representations is postulated as the basis for functional recovery following many types of nervous system injury. Neuronal mechanisms underlying this form of cortical plasticity are poorly understood. The present study investigated the hypothesis that the basal forebrain cholinergic system plays an essential role in enabling the cortical reorganization required for functional recovery following brain injury. The results demonstrate that functional recovery following cortical injury requires basal forebrain cholinergic mechanisms and suggest that the basis for this recovery is the cholinergic-dependent reorganization of motor representations. These findings raise the intriguing possibility that deficits in cholinergic function may limit functional outcomes following nervous system injury.

Intraspinal and Intracortical Delivery of AAV Vectors for Intersectional Circuit Tracing in Non-transgenic Species.

The mapping of long-range axonal projections is a rapidly growing endeavor in the field of neuroscience. Recent advances in the development of adeno-associated viral vectors that can achieve efficient retrograde transport now enable the characterization and manipulation of specific neuronal subpopulations using Cre-dependent, intersectional approaches. Importantly, these approaches can be applied to non-transgenic animals, making it possible to carry out detailed anatomical studies across a variety of species including nonhuman primates. In this chapter, we demonstrate the utility of such intersectional strategies by describing methods for targeting viral constructs to distinct subsets of corticospinal motor neurons based on their projections to specific spinal cord segments.

Lesions of the Basal forebrain cholinergic system impair task acquisition and abolish cortical plasticity associated with motor skill learning.

The contribution of the basal forebrain cholinergic system in mediating plasticity of cortical sensorimotor representations was examined in the context of normal learning. The effects of specific basal forebrain cholinergic lesions upon cortical reorganization associated with learning a skilled motor task were investigated, addressing, for the first time, the functional consequences of blocking cortical map plasticity. Results demonstrate that disrupting basal forebrain cholinergic function disrupts cortical map reorganization and impairs motor learning. Cholinergic lesions do not impair associative fear learning or overall sensorimotor function. These results support the hypothesis that the basal forebrain cholinergic system may be specifically implicated in forms of learning requiring plasticity of cortical representations.

Thalamocortical Projections onto Behaviorally Relevant Neurons Exhibit Plasticity during Adult Motor Learning.

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.

Nerve Growth Factor Gene Therapy: Activation of Neuronal Responses in Alzheimer Disease.

IMPORTANCE: Alzheimer disease (AD) is the most common neurodegenerative disorder and lacks effective disease-modifying therapies. In 2001, we initiated a clinical trial of nerve growth factor (NGF) gene therapy in AD, the first effort at gene delivery in an adult neurodegenerative disorder. This program aimed to determine whether a nervous system growth factor prevents or reduces cholinergic neuronal degeneration in patients with AD. We present postmortem findings in 10 patients with survival times ranging from 1 to 10 years after treatment. OBJECTIVE: To determine whether degenerating neurons in AD retain an ability to respond to a nervous system growth factor delivered after disease onset. DESIGN, SETTING, AND PARTICIPANTS: Patients in this anatomicopathological study were enrolled in clinical trials from March 2001 to October 2012 at the University of California, San Diego, Medical Center in La Jolla. Ten patients with early AD underwent NGF gene therapy using ex vivo or in vivo gene transfer. The brains of all 8 patients in the first phase 1 ex vivo trial and of 2 patients in a subsequent phase 1 in vivo trial were examined. MAIN OUTCOMES AND MEASURES: Brains were immunolabeled to evaluate in vivo gene expression, cholinergic neuronal responses to NGF, and activation of NGF-related cell signaling. In 2 patients, NGF protein levels were measured by enzyme-linked immunosorbent assay. RESULTS: Among 10 patients, degenerating neurons in the AD brain responded to NGF. All patients exhibited a trophic response to NGF in the form of axonal sprouting toward the NGF source. Comparing treated and nontreated sides of the brain in 3 patients who underwent unilateral gene transfer, cholinergic neuronal hypertrophy occurred on the NGF-treated side (P < .05). Activation of cellular signaling and functional markers was present in 2 patients who underwent adeno-associated viral vectors (serotype 2)-mediated NGF gene transfer. Neurons exhibiting tau pathology and neurons free of tau expressed NGF, indicating that degenerating cells can be infected with therapeutic genes, with resultant activation of cell signaling. No adverse pathological effects related to NGF were observed. CONCLUSIONS AND RELEVANCE: These findings indicate that neurons of the degenerating brain retain the ability to respond to growth factors with axonal sprouting, cell hypertrophy, and activation of functional markers. Sprouting induced by NGF persists for 10 years after gene transfer. Growth factor therapy appears safe over extended periods and merits continued testing as a means of treating neurodegenerative disorders.

Nerve growth factor, neuropeptides, and mast cells in ultraviolet-B-induced systemic suppression of contact hypersensitivity responses in mice.

The induction of systemic immunosuppression following ultraviolet B radiation exposure has been linked with the release of inflammatory and immunomodulatory mediators by cells of the epidermis and dermis. Nerve growth factor has not previously been linked with ultraviolet-B-induced immunosuppressive effects. Nerve growth factor antibodies abrogated ultraviolet-B-induced systemic suppression of contact hypersensitivity responses in BALB/C mice. Subcutaneous injection of nerve growth factor (20 microg per mouse) into dorsal skin 5 d before hapten sensitization on ventral skin suppressed contact hypersensitivity responses in mast-cell-replete but not Wf/Wf mast-cell-depleted mice. Nerve growth factor injected 24 h prior to challenge was not able to suppress the efferent phase of the contact hypersensitivity response. Subcutaneous injection of nerve growth factor (20 microg per mouse) did not suppress contact hypersensitivity responses in capsaicin-pretreated (neuropeptide-depleted) BALB/c mice, and thus sensory c-fibers are necessary for nerve-growth-factor-mediated systemic suppression of contact hypersensitivity responses. Increased concentrations of nerve growth factor within epidermal keratinocytes 8 h after ultraviolet B irradiation were confirmed immunohistochemically. These findings support a role for keratinocyte-derived nerve growth factor via its action on sensory c-fibers, and subsequent release of neuropeptides to mediate mast cell degranulation in systemic suppression of contact hypersensitivity responses in mice following ultraviolet B exposure.

Nerve growth factor is primarily produced by GABAergic neurons of the adult rat cortex.

Within the cortex, nerve growth factor (NGF) mediates the innervation of cholinergic neurons during development, maintains cholinergic corticopetal projections during adulthood and modulates cholinergic function through phenotypic control of the cholinergic gene locus. Recent studies suggest NGF may also play an important role in cortical plasticity in adulthood. Previously, NGF-producing cells have been shown to colocalize with GABAergic cell markers within the hippocampus, striatum, and basal forebrain. Classification of cells producing NGF in the cortex is lacking, however, and cholinergic corticopetal projections have been shown to innervate both pyramidal and GABAergic neurons in the cortex. In order to clarify potential trophic interactions between cortical neurons and cholinergic projections, we used double-fluorescent immunohistochemistry to classify NGF-expressing cells in several cortical regions, including the prefrontal cortex, primary motor cortex, parietal cortex and temporal cortex. Our results show that NGF colocalizes extensively with GABAergic cell markers in all cortical regions examined, with >91% of NGF-labeled cells coexpressing GAD65/67. Conversely, NGF-labeled cells exhibit very little co-localization with the excitatory cell marker CaMKIIα (<5% of cells expressing NGF). NGF expression was present in 56% of GAD-labeled cells, suggesting that production is confined to a specific subset of GABAergic neurons. These findings demonstrate that GABAergic cells are the primary source of NGF production in the cortex, and likely support the maintenance and function of basal forebrain cholinergic projections in adulthood.

Neural stem cells in models of spinal cord injury.

Replication of published studies is an important and respected aspect of the conduct of science. Most would argue that the interpretation of "negative" outcomes is still more challenging than the interpretation of "positive" findings, however, due to uncertainty in knowing precisely why a hypothesized outcome was not observed: in particular, are "negative" findings in replication studies a result of invalidity of the original experimental hypothesis, or due to a methodological failure, insensitivity of the applied instruments of analysis, or other factors? These points must be carefully considered. Steward and colleagues report findings of a study in which multipotent neural progenitor cells were grafted to sites of T3 complete transection. Unlike our study, cells failed to fill the lesion site, leaving collagenous rifts between rostral and caudal graft components. This "anatomical" failure precluded formation of neural relays across the lesion site, and was predictably associated with a failure to detect functional improvement. In summarizing outcomes of the study, Steward and colleagues did not clearly link the failure to achieve graft continuity in the lesion cavity with functional outcomes, despite the central role of this observation in cogently interpreting results of the replication study. In addition, the authors stated that they failed to replicate our report of "extensive" host axonal regeneration into grafts, but we did not report "extensive" host anatomical regeneration; moreover, underexposed images may have contributed to Steward's underestimation of host axonal penetration. The authors also stated that our original study excluded some animals from functional analysis, and this is incorrect. While replication studies are important and necessary, this particular report contained several errors, and the failure to form a continuous neural progenitor cell bridge across the lesion site limited the ability to conclude whether continuous grafts can restore function. In subsequent experiments we too have observed rift formation in animals grafted at long delays (>2weeks) after SCI, and we confirm that animals with rifts do not exhibit functional improvement; we are developing methods to remove or prevent rift formation. The replication study confirmed the cardinal finding of our original report: that early-stage neural precursors extend very large numbers of axons over remarkably long distances through the lesioned adult spinal cord.

Templated agarose scaffolds for the support of motor axon regeneration into sites of complete spinal cord transection.

Bioengineered scaffolds have the potential to support and guide injured axons after spinal cord injury, contributing to neural repair. In previous studies we have reported that templated agarose scaffolds can be fabricated into precise linear arrays and implanted into the partially injured spinal cord, organizing growth and enhancing the distance over which local spinal cord axons and ascending sensory axons extend into a lesion site. However, most human injuries are severe, sparing only thin rims of spinal cord tissue in the margins of a lesion site. Accordingly, in the present study we examined whether template agarose scaffolds seeded with bone marrow stromal cells secreting Brain-Derived Neurotrophic Factor (BDNF) would support regeneration into severe, complete spinal cord transection sites. Moreover, we tested responses of motor axon populations originating from the brainstem. We find that templated agarose scaffolds support motor axon regeneration into a severe spinal cord injury model and organize axons into fascicles of highly linear configuration. BDNF significantly enhances axonal growth. Collectively, these findings support the feasibility of scaffold implantation for enhancing central regeneration after even severe central nervous system injury.