Basal ganglia-spinal cord pathway that commands locomotor gait asymmetries in mice.

The basal ganglia are essential for executing motor actions. How the basal ganglia engage spinal motor networks has remained elusive. Medullary Chx10 gigantocellular (Gi) neurons are required for turning gait programs, suggesting that turning gaits organized by the basal ganglia are executed via this descending pathway. Performing deep brainstem recordings of Chx10 Gi Ca2+ activity in adult mice, we show that striatal projection neurons initiate turning gaits via a dominant crossed pathway to Chx10 Gi neurons on the contralateral side. Using intersectional viral tracing and cell-type-specific modulation, we uncover the principal basal ganglia-spinal cord pathway for locomotor asymmetries in mice: basal ganglia → pontine reticular nucleus, oral part (PnO) → Chx10 Gi → spinal cord. Modulating the restricted PnO → Chx10 Gi pathway restores turning competence upon striatal damage, suggesting that dysfunction of this pathway may contribute to debilitating turning deficits observed in Parkinson's disease. Our results reveal the stratified circuit architecture underlying a critical motor program.

Brainstem Circuits for Locomotion.

Locomotion is a universal motor behavior that is expressed as the output of many integrated brain functions. Locomotion is organized at several levels of the nervous system, with brainstem circuits acting as the gate between brain areas regulating innate, emotional, or motivational locomotion and executive spinal circuits. Here we review recent advances on brainstem circuits involved in controlling locomotion. We describe how delineated command circuits govern the start, speed, stop, and steering of locomotion. We also discuss how these pathways interface between executive circuits in the spinal cord and diverse brain areas important for context-specific selection of locomotion. A recurrent theme is the need to establish a functional connectome to and from brainstem command circuits. Finally, we point to unresolved issues concerning the integrated function of locomotor control.

Brainstem neurons that command mammalian locomotor asymmetries.

Descending command neurons instruct spinal networks to execute basic locomotor functions, such as gait and speed. The command functions for gait and speed are symmetric, implying that a separate unknown system directs asymmetric movements, including the ability to move left or right. In the present study, we report that Chx10-lineage reticulospinal neurons act to control the direction of locomotor movements in mammals. Chx10 neurons exhibit mainly ipsilateral projection, and their selective unilateral activation causes ipsilateral turning movements in freely moving mice. Unilateral inhibition of Chx10 neurons causes contralateral turning movements. Paired left-right motor recordings identified distinct mechanisms for directional movements mediated via limb and axial spinal circuits. Finally, we identify sensorimotor brain regions that project on to Chx10 reticulospinal neurons, and demonstrate that their unilateral activation can impart left-right directional commands. Together these data identify the descending motor system that commands left-right locomotor asymmetries in mammals.

Phrenic-specific transcriptional programs shape respiratory motor output.

The precise pattern of motor neuron (MN) activation is essential for the execution of motor actions; however, the molecular mechanisms that give rise to specific patterns of MN activity are largely unknown. Phrenic MNs integrate multiple inputs to mediate inspiratory activity during breathing and are constrained to fire in a pattern that drives efficient diaphragm contraction. We show that Hox5 transcription factors shape phrenic MN output by connecting phrenic MNs to inhibitory premotor neurons. Hox5 genes establish phrenic MN organization and dendritic topography through the regulation of phrenic-specific cell adhesion programs. In the absence of Hox5 genes, phrenic MN firing becomes asynchronous and erratic due to loss of phrenic MN inhibition. Strikingly, mice lacking Hox5 genes in MNs exhibit abnormal respiratory behavior throughout their lifetime. Our findings support a model where MN-intrinsic transcriptional programs shape the pattern of motor output by orchestrating distinct aspects of MN connectivity.

In mammals, air is moved in and out of the lungs by a sheet of muscle called the diaphragm. When this muscle contracts air gets drawn into the lungs and as the muscle relaxes this pushes air back out. Movement of the diaphragm is controlled by a group of nerve cells called motor neurons which are part of the phrenic motor column (or PMC for short) that sits within the spinal cord. The neurons within this column work together with nerve cells in the brain to coordinate the speed and duration of each breath. For the lungs to develop normally, the neurons that control how the diaphragm contracts need to start working before birth. During development, motor neurons in the PMC cluster together and connect with other nerve cells involved in breathing. But, despite their essential role, it is not yet clear how neurons in the PMC develop and join up with other nerve cells. Now, Vagnozzi et al. show that a set of genes which make the transcription factor Hox5 control the position and organization of motor neurons in the PMC. Transcription factors work as genetic switches, turning sets of genes on and off. Vagnozzi et al. showed that removing the Hox5 transcription factors from motor neurons in the PMC changed their activity and disordered their connections with other breathing-related nerve cells. Hox5 transcription factors regulate the production of proteins called cadherins which join together neighboring cells. Therefore, motor neurons lacking Hox5 were unable to make enough cadherins to securely stick together and connect with other nerve cells. Further experiments showed that removing the genes that code for Hox5 caused mice to have breathing difficulties in the first two weeks after birth. Although half of these mutant mice were eventually able to breathe normally, the other half died within a week. These breathing defects are reminiscent of the symptoms observed in sudden infant death syndrome (also known as SIDS). Abnormalities in breathing occur in many other diseases, including sleep apnea, muscular dystrophy and amyotrophic lateral sclerosis (ALS). A better understanding of how the connections between nerve cells involved in breathing are formed, and the role of Hox5 and cadherins, could lead to improved treatment options for these diseases.

Rapid functional genetics of the oligodendrocyte lineage using pluripotent stem cells.

Oligodendrocyte dysfunction underlies many neurological disorders, but rapid assessment of mutation-specific effects in these cells has been impractical. To enable functional genetics in oligodendrocytes, here we report a highly efficient method for generating oligodendrocytes and their progenitors from mouse embryonic and induced pluripotent stem cells, independent of mouse strain or mutational status. We demonstrate that this approach, when combined with genome engineering, provides a powerful platform for the expeditious study of genotype-phenotype relationships in oligodendrocytes.

Phasic inhibition as a mechanism for generation of rapid respiratory rhythms.

Central neural networks operate continuously throughout life to control respiration, yet mechanisms regulating ventilatory frequency are poorly understood. Inspiration is generated by the pre-Bötzinger complex of the ventrolateral medulla, where it is thought that excitation increases inspiratory frequency and inhibition causes apnea. To test this model, we used an in vitro optogenetic approach to stimulate select populations of hindbrain neurons and characterize how they modulate frequency. Unexpectedly, we found that inhibition was required for increases in frequency caused by stimulation of Phox2b-lineage, putative CO2-chemosensitive neurons. As a mechanistic explanation for inhibition-dependent increases in frequency, we found that phasic stimulation of inhibitory neurons can increase inspiratory frequency via postinhibitory rebound. We present evidence that Phox2b-mediated increases in frequency are caused by rebound excitation following an inhibitory synaptic volley relayed by expiration. Thus, although it is widely thought that inhibition between inspiration and expiration simply prevents activity in the antagonistic phase, we instead propose a model whereby inhibitory coupling via postinhibitory rebound excitation actually generates fast modes of inspiration.

A Latent Propriospinal Network Can Restore Diaphragm Function after High Cervical Spinal Cord Injury.

Spinal cord injury (SCI) above cervical level 4 disrupts descending axons from the medulla that innervate phrenic motor neurons, causing permanent paralysis of the diaphragm. Using an ex vivo preparation in neonatal mice, we have identified an excitatory spinal network that can direct phrenic motor bursting in the absence of medullary input. After complete cervical SCI, blockade of fast inhibitory synaptic transmission caused spontaneous, bilaterally coordinated phrenic bursting. Here, spinal cord glutamatergic neurons were both sufficient and necessary for the induction of phrenic bursts. Direct stimulation of phrenic motor neurons was insufficient to evoke burst activity. Transection and pharmacological manipulations showed that this spinal network acts independently of medullary circuits that normally generate inspiration, suggesting a distinct non-respiratory function. We further show that this "latent" network can be harnessed to restore diaphragm function after high cervical SCI in adult mice and rats.

Overexpression of the monocyte chemokine CCL2 in dorsal root ganglion neurons causes a conditioning-like increase in neurite outgrowth and does so via a STAT3 dependent mechanism.

Neuroinflammation plays a critical role in the regeneration of peripheral nerves following axotomy. An injury to the sciatic nerve leads to significant macrophage accumulation in the L5 DRG, an effect not seen when the dorsal root is injured. We recently demonstrated that this accumulation around axotomized cell bodies is necessary for a peripheral conditioning lesion response to occur. Here we asked whether overexpression of the monocyte chemokine CCL2 specifically in DRG neurons of uninjured mice is sufficient to cause macrophage accumulation and to enhance regeneration or whether other injury-derived signals are required. AAV5-EF1α-CCL2 was injected intrathecally, and this injection led to a time-dependent increase in CCL2 mRNA expression and macrophage accumulation in L5 DRG, with a maximal response at 3 weeks post-injection. These changes led to a conditioning-like increase in neurite outgrowth in DRG explant and dissociated cell cultures. This increase in regeneration was dependent upon CCL2 acting through its primary receptor CCR2. When CCL2 was overexpressed in CCR2-/- mice, macrophage accumulation and enhanced regeneration were not observed. To address the mechanism by which CCL2 overexpression enhances regeneration, we tested for elevated expression of regeneration-associated genes in these animals. Surprisingly, we found that CCL2 overexpression led to a selective increase in LIF mRNA and neuronal phosphorylated STAT3 (pSTAT3) in L5 DRGs, with no change in expression seen in other RAGs such as GAP-43. Blockade of STAT3 phosphorylation by each of two different inhibitors prevented the increase in neurite outgrowth. Thus, CCL2 overexpression is sufficient to induce macrophage accumulation in uninjured L5 DRGs and increase the regenerative capacity of DRG neurons via a STAT3-dependent mechanism.

Modulation of the proteoglycan receptor PTPσ promotes recovery after spinal cord injury.

Contusive spinal cord injury leads to a variety of disabilities owing to limited neuronal regeneration and functional plasticity. It is well established that an upregulation of glial-derived chondroitin sulphate proteoglycans (CSPGs) within the glial scar and perineuronal net creates a barrier to axonal regrowth and sprouting. Protein tyrosine phosphatase σ (PTPσ), along with its sister phosphatase leukocyte common antigen-related (LAR) and the nogo receptors 1 and 3 (NgR), have recently been identified as receptors for the inhibitory glycosylated side chains of CSPGs. Here we find in rats that PTPσ has a critical role in converting growth cones into a dystrophic state by tightly stabilizing them within CSPG-rich substrates. We generated a membrane-permeable peptide mimetic of the PTPσ wedge domain that binds to PTPσ and relieves CSPG-mediated inhibition. Systemic delivery of this peptide over weeks restored substantial serotonergic innervation to the spinal cord below the level of injury and facilitated functional recovery of both locomotor and urinary systems. Our results add a new layer of understanding to the critical role of PTPσ in mediating the growth-inhibited state of neurons due to CSPGs within the injured adult spinal cord.

Functional regeneration beyond the glial scar.

Astrocytes react to CNS injury by building a dense wall of filamentous processes around the lesion. Stromal cells quickly take up residence in the lesion core and synthesize connective tissue elements that contribute to fibrosis. Oligodendrocyte precursor cells proliferate within the lesion and entrap dystrophic axon tips. Here we review evidence that this aggregate scar acts as the major barrier to regeneration of axons after injury. We also consider several exciting new interventions that allow axons to regenerate beyond the glial scar, and discuss the implications of this work for the future of regeneration biology.

Robust CNS regeneration after complete spinal cord transection using aligned poly-L-lactic acid microfibers.

Following spinal cord injury, axons fail to regenerate without exogenous intervention. In this study we report that aligned microfiber-based grafts foster robust regeneration of vascularized CNS tissue. Film, random, and aligned microfiber-based conduits were grafted into a 3 mm thoracic rat spinal cord gap created by complete transection. Over the course of 4 weeks, microtopography presented by aligned or random poly-L-lactic acid microfibers facilitated infiltration of host tissue, and the initial 3 mm gap was closed by endogenous cell populations. This bulk tissue response was composed of regenerating axons accompanied by morphologically aligned astrocytes. Aligned fibers promoted long distance (2055 ± 150 µm), rostrocaudal axonal regeneration, significantly greater than random fiber (1162 ± 87 µm) and film (413 ± 199 µm) controls. Retrograde tracing indicated that regenerating axons originated from propriospinal neurons of the rostral spinal cord, and supraspinal neurons of the reticular formation, red nucleus, raphe and vestibular nuclei. Our findings outline a form of regeneration within the central nervous system that holds important implications for regeneration biology.

Varying the diameter of aligned electrospun fibers alters neurite outgrowth and Schwann cell migration.

Aligned, electrospun fibers have shown great promise in facilitating directed neurite outgrowth within cell and animal models. While electrospun fiber diameter does influence cellular behavior, it is not known how aligned, electrospun fiber scaffolds of differing diameter influence neurite outgrowth and Schwann cell (SC) migration. Thus, the goal of this study was to first create highly aligned, electrospun fiber scaffolds of varying diameter and then assess neurite and SC behavior from dorsal root ganglia (DRG) explants. Three groups of highly aligned, electrospun poly-l-lactic acid (PLLA) fibers were created (1325+383 nm, large diameter fibers; 759+179 nm, intermediate diameter fibers; and 293+65 nm, small diameter fibers). Embryonic stage nine (E9) chick DRG were cultured on fiber substrates for 5 days and then the explants were stained against neurofilament and S100. DAPI stain was used to assess SC migration. Neurite length and SC migration distance were determined. In general, the direction of neurite extension and SC migration were guided along the aligned fibers. On the small diameter fiber substrate, the neurite length was 42% and 36% shorter than those on the intermediate and large fiber substrates, respectively. Interestingly, SC migration did not correlate with that of neurite extension in all situations. SCs migrated equivalently with extending neurites in both the small and large diameter scaffolds, but lagged behind neurites on the intermediate diameter scaffolds. Thus, in some situations, topography alone is sufficient to guide neurites without the leading support of SCs. Scanning electron microscopy images show that neurites cover the fibers and do not reside exclusively between fibers. Further, at the interface between fibers and neurites, filopodial extensions grab and attach to nearby fibers as they extend down the fiber substrate. Overall, the results and observations suggest that fiber diameter is an important parameter to consider when constructing aligned, electrospun fibers for nerve regeneration applications.

A rapid, quantitative method for assessing axonal extension on biomaterial platforms.

Measuring outgrowth of neuronal explants is critical in evaluating the ability of a biomaterial to act as a permissive substrate for neuronal adhesion and growth. Previous methods lack the ability to quantify robust outgrowth, or lack the capacity to quantify growth on opaque substrates because they exploit the transparent nature of culture dishes to segregate neuronal processes from an image background based on color intensity. In this study, we sought to investigate the ability of opaque silica sol-gel materials to facilitate axonal outgrowth; therefore, a method was developed for quantifying outgrowth of neurites from dorsal root ganglion explants on these unique surfaces. Dorsal root ganglia were isolated from stage-nine chick embryos and cultured for 48 h on sol-gel materials presenting agarose and chitosan polysaccharides individually or in combination. Explants were then imaged, and basic image analysis software was used by three independent observers to obtain axonal length and axonal area measurements. Robust axon length and axonal spread measurements for ganglia cultured on agarose-chitosan sol-gel matrices yield an estimate of strong neural compatibility for these substrates over silica matrices presenting no polysaccharides, or silica matrices presenting chitosan or agarose individually. We suggest that this simple protocol for quantifying material biocompatibility offers an analysis strategy that can be used universally to the same end.

Creation of highly aligned electrospun poly-L-lactic acid fibers for nerve regeneration applications.

Aligned, electrospun polymer fibers have shown considerable promise in directing regenerating axons in vitro and in vivo. However, in several studies, final electrospinning parameters are presented for producing aligned fiber scaffolds, and alignment where minimal fiber crossing occurs is not achieved. Highly aligned species are necessary for neural tissue engineering applications to ensure that axonal extension occurs through a regenerating environment efficiently. Axonal outgrowth on fibers that deviate from the natural axis of growth may delay axonal extension from one end of a scaffold to the other. Therefore, producing aligned fiber scaffolds with little fiber crossing is essential. In this study, the contributions of four electrospinning parameters (collection disk rotation speed, needle size, needle tip shape and syringe pump flow rate) were investigated thoroughly with the goal of finding parameters to obtain highly aligned electrospun fibers made from poly-L-lactic acid (PLLA). Using an 8 wt% PLLA solution in chloroform, a collection disk rotation speed of 1000 revolutions per minute (rpm), a 22 gauge, sharp-tip needle and a syringe pump rate of 2 ml h(-1) produced highly aligned fiber (1.2-1.6 microm in diameter) scaffolds verified using a fast Fourier transform and a fiber alignment quantification technique. Additionally, the application of an insulating sheath around the needle tip improved the rate of fiber deposition (electrospinning efficiency). Optimized scaffolds were then evaluated in vitro using embryonic stage nine (E9) chick dorsal root ganglia (DRGs) and rat Schwann cells (SCs). To demonstrate the importance of creating highly aligned scaffolds to direct neurite outgrowth, scaffolds were created that contained crossing fibers. Neurites on these scaffolds were directed down the axis of the aligned fibers, but neurites also grew along the crossed fibers. At times, these crossed fibers even stopped further axonal extension. Highly aligned PLLA fibers generated under optimized electrospinning conditions guided neurite and SC growth along the aligned fibers. Schwann cells demonstrated the bipolar phenotype seen along the fibers. Using a novel technique to determine fiber density, an increase in fiber density correlated to an increase in the number of neurites, but average neurite length was not statistically different between the two different fiber densities. Together, this work presents methods by which to produce highly aligned fiber scaffolds efficiently and techniques for assessing neurite outgrowth on different fiber scaffolds, while suggesting that crossing fibers may be detrimental in fostering efficient, directed axonal outgrowth.