Catenin signaling controls phrenic motor neuron development and function during a narrow temporal window.

Phrenic Motor Column (PMC) neurons are a specialized subset of motor neurons (MNs) that provide the only motor innervation to the diaphragm muscle and are therefore essential for survival. Despite their critical role, the mechanisms that control phrenic MN development and function are not well understood. Here, we show that catenin-mediated cadherin adhesive function is required for multiple aspects of phrenic MN development.

Catenin signaling controls phrenic motor neuron development and function during a narrow temporal window.

Phrenic Motor Column (PMC) neurons are a specialized subset of motor neurons (MNs) that provide the only motor innervation to the diaphragm muscle and are therefore essential for survival. Despite their critical role, the mechanisms that control phrenic MN development and function are not well understood. Here, we show that catenin-mediated cadherin adhesive function is required for multiple aspects of phrenic MN development.

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.

General principles of spinal motor circuit development: early contributions from research on avian embryos.

Birds and mammals, both being amniotes, share many common aspects of development. Thus our understanding of how limb-innervating mammalian spinal motor circuits develop was greatly influenced by the use of the avian embryo (chick/quail) to bring about experimental perturbations to identify basic underlying mechanisms. These included embryonic surgery, the application of drugs to influence activity or molecular interactions, and the ability to observe motor behavior and make physiological recordings in intact developing embryos.

Distinct Roles of Different Presynaptic and Postsynaptic NCAM Isoforms in Early Motoneuron-Myotube Interactions Required for Functional Synapse Formation.

The neural cell adhesion molecule (NCAM) is expressed both presynaptically and postsynaptically during neuromuscular junction formation. Genetic deletion in mice of all three isoforms (180, 140, and 120 kDa), or just the 180 isoform, suggested that different isoforms played distinct roles in synaptic maturation. Here we characterized in mice of either sex the earliest adhesive contacts between the growth cones of motoneurons and myotubes and their subsequent maturation into functional synapses in cocultures of motoneurons and myotubes, which expressed their normal complement of NCAM isoforms, or were lacking all isoforms either presynaptically or postsynaptically.

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.

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.

Optogenetic-mediated increases in in vivo spontaneous activity disrupt pool-specific but not dorsal-ventral motoneuron pathfinding.

Rhythmic waves of spontaneous electrical activity are widespread in the developing nervous systems of birds and mammals, and although many aspects of neural development are activity-dependent, it has been unclear if rhythmic waves are required for in vivo motor circuit development, including the proper targeting of motoneurons to muscles. We show here that electroporated channelrhodopsin-2 can be activated in ovo with light flashes to drive waves at precise intervals of approximately twice the control frequency in intact chicken embryos. Optical monitoring of associated axial movements ensured that the altered frequency was maintained.

Frequency-dependent modes of synaptic vesicle endocytosis and exocytosis at adult mouse neuromuscular junctions.

During locomotion, adult rodent lumbar motoneurons fire in high-frequency (80-100 Hz) 1-2 s bursts every several seconds, releasing between 10,000 and 20,000 vesicles per burst. The estimated total vesicle pool size indicates that all vesicles would be used within 30 s; thus, a mechanism for rapid endocytosis and vesicle recycling is necessary to maintain effective transmission and motor behavior. However, whether such rapid recycling exists at mouse neuromuscular junctions (NMJs) or how it is regulated has been unclear.

Reduced survival of motor neuron (SMN) protein in motor neuronal progenitors functions cell autonomously to cause spinal muscular atrophy in model mice expressing the human centromeric (SMN2) gene.

Spinal muscular atrophy (SMA) is a common (approximately 1:6400) autosomal recessive neuromuscular disorder caused by a paucity of the survival of motor neuron (SMN) protein. Although widely recognized to cause selective spinal motor neuron loss when deficient, the precise cellular site of action of the SMN protein in SMA remains unclear. In this study we sought to determine the consequences of selectively depleting SMN in the motor neurons of model mice.

In vivo activation of channelrhodopsin-2 reveals that normal patterns of spontaneous activity are required for motoneuron guidance and maintenance of guidance molecules.

Spontaneous, highly rhythmic episodes of propagating bursting activity are present early during the development of chick and mouse spinal cords. Acetylcholine, and GABA and glycine, which are both excitatory at this stage, provide the excitatory drive. It was previously shown that a moderate decrease in the frequency of bursting activity, caused by in ovo application of the GABA(A) receptor blocker, picrotoxin, resulted in motoneurons making dorsal-ventral (D-V) pathfinding errors in the limb and in the altered expression of guidance molecules associated with this decision.

Serotonergic neurons migrate radially through the neuroepithelium by dynamin-mediated somal translocation.

Embryonic CNS neurons can migrate from the ventricular zone to their final destination by radial glial-guided locomotion. Another less appreciated mechanism is somal translocation, where the young neuron maintains its primitive ventricular and pial processes, through which the cell body moves. A major problem in studying translocation has been the identification of neuronal-specific markers that appear in primitive, radially shaped cells.

Characterization of rhythmic Ca2+ transients in early embryonic chick motoneurons: Ca2+ sources and effects of altered activation of transmitter receptors.

In the nervous system, spontaneous Ca(2+) transients play important roles in many developmental processes. We previously found that altering the frequency of electrically recorded rhythmic spontaneous bursting episodes in embryonic chick spinal cords differentially perturbed the two main pathfinding decisions made by motoneurons, dorsal-ventral and pool-specific, depending on the sign of the frequency alteration. Here, we characterized the Ca(2+) transients associated with these bursts and showed that at early stages while motoneurons are still migrating and extending axons to the base of the limb bud, they display spontaneous, highly rhythmic, and synchronized Ca(2+) transients.

Reduced SMN protein impairs maturation of the neuromuscular junctions in mouse models of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a common pediatric neuromuscular disorder caused by insufficient levels of the survival of motor neuron (SMN) protein. Studies involving SMA patients and animal models expressing the human SMN2 gene have yielded relatively little information about the earliest cellular consequences of reduced SMN protein. In this study, we have used severe- and mild-SMN2 expressing mouse models of SMA as well as material from human patients to understand the initial stages of neurodegeneration in the human disease.

Selective targeting of different neural cell adhesion molecule isoforms during motoneuron myotube synapse formation in culture and the switch from an immature to mature form of synaptic vesicle cycling.

Characterization of neuromuscular junction formation and function in mice lacking all neural cell adhesion molecule (NCAM) isoforms or only the 180 isoform demonstrated that the 180 isoform was required at adult synapses to maintain effective transmission with repetitive stimulation whereas the 140 and/or 120 isoform(s) were sufficient to mediate the downregulation of synaptic vesicle cycling along the axon after synapse formation. However, the expression and targeting of each isoform and its relationship to distinct forms of synaptic vesicle cycling before and after synapse formation was previously unknown. By transfecting chick motoneurons with fluorescently tagged mouse 180, 140 and 120 isoforms, we show that before myotube contact the 180 and 140 isoforms are expressed in distinct puncta along the axon which are sites of an immature form (Brefeldin A sensitive, L-type Ca2+ channel mediated) of vesicle cycling.

Spontaneous rhythmic activity in early chick spinal cord influences distinct motor axon pathfinding decisions.

During embryonic development chick and mouse spinal cords are activated by highly rhythmic episodes of spontaneous bursting activity at very early stages, while motoneurons are still migrating and beginning to extend their axons to the base of the limb. While such spontaneous activity has been shown to be important in refining neural projections once axons have reached their targets, early pathfinding events have been thought to be activity independent. However, in-ovo pharmacological manipulation of the transmitter systems that drive such early activity has shown that early motor axon pathfinding events are highly dependent on the normal pattern of bursting activity.

New optical tools for controlling neuronal activity.

A major challenge in understanding the relationship between neural activity and development, and ultimately behavior, is to control simultaneously the activity of either many neurons belonging to specific subsets or specific regions within individual neurons. Optimally, such a technique should be capable of both switching nerve cells on and off within milliseconds in a non-invasive manner, and inducing depolarizations or hyperpolarizations for periods lasting from milliseconds to many seconds. Specific ion conductances in subcellular compartments must also be controlled to bypass signaling cascades in order to regulate precisely cellular events such as synaptic transmission.

Increasing the frequency of spontaneous rhythmic activity disrupts pool-specific axon fasciculation and pathfinding of embryonic spinal motoneurons.

Rhythmic spontaneous bursting activity, which occurs in many developing neural circuits, has been considered to be important for the refinement of neural projections but not for early pathfinding decisions. However, the precise frequency of bursting activity differentially affects the two major pathfinding decisions made by chick lumbosacral motoneurons. Moderate slowing of burst frequency was shown previously to cause motoneurons to make dorsoventral (D-V) pathfinding errors and to alter the expression of molecules involved in that decision.

CD24 is expressed by myofiber synaptic nuclei and regulates synaptic transmission.

The genes encoding several synaptic proteins, including acetylcholine receptors, acetylcholinesterase, and the muscle-specific kinase, MuSK, are expressed selectively by a small number of myofiber nuclei positioned near the synaptic site. Genetic analysis of mutant mice suggests that additional genes, expressed selectively by synaptic nuclei, might encode muscle-derived retrograde signals that regulate the differentiation of motor axon terminals. To identify candidate retrograde signals, we used a microarray screen to identify genes that are preferentially expressed in the synaptic region of muscle, and we analyzed one such gene, CD24, further.

Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin.

Techniques for fast noninvasive control of neuronal excitability will be of major importance for analyzing and understanding neuronal networks and animal behavior. To develop these tools we demonstrated that two light-activated signaling proteins, vertebrate rat rhodopsin 4 (RO4) and the green algae channelrhodospin 2 (ChR2), could be used to control neuronal excitability and modulate synaptic transmission. Vertebrate rhodopsin couples to the Gi/o, pertussis toxin-sensitive pathway to allow modulation of G protein-gated inward rectifying potassium channels and voltage-gated Ca2+ channels.

NCAM 180 acting via a conserved C-terminal domain and MLCK is essential for effective transmission with repetitive stimulation.

NCAM 180 isoform null neuromuscular junctions are unable to effectively mobilize and exocytose synaptic vesicles and thus exhibit periods of total transmission failure during high-frequency repetitive stimulation. We have identified a highly conserved C-terminal (KENESKA) domain on NCAM that is required to maintain effective transmission and demonstrate that it acts via a pathway involving MLCK and probably myosin light chain (MLC) and myosin II. By perfecting a method of introducing peptides into adult NMJs, we tested the hypothesized role of proteins in this pathway by competitive disruption of protein-protein interactions.

Cholinergic input is required during embryonic development to mediate proper assembly of spinal locomotor circuits.

Rhythmic limb movements are controlled by pattern-generating neurons within the ventral spinal cord, but little is known about how these locomotor circuits are assembled during development. At early stages of embryogenesis, motor neurons are spontaneously active, releasing acetylcholine that triggers the depolarization of adjacent cells in the spinal cord. To investigate whether acetylcholine-driven activity is required for assembly of the central pattern-generating (CPG) circuit, we studied mice lacking the choline acetyltransferase (ChAT) enzyme.

Adrenal chromaffin cells exhibit impaired granule trafficking in NCAM knockout mice.

Neural cell adhesion molecule (NCAM) plays several critical roles in neuron path-finding and intercellular communication during development. In the clinical setting, serum NCAM levels are altered in both schizophrenic and autistic patients. NCAM knockout mice have been shown to exhibit deficits in neuronal functions including impaired hippocampal long term potentiation and motor coordination.

Normal patterns of spontaneous activity are required for correct motor axon guidance and the expression of specific guidance molecules.

Rhythmic spontaneous electrical activity occurs in many parts of the developing nervous system, where it plays essential roles in the refinement of neural connections. By blocking or slowing this bursting activity, via in ovo drug applications at precise developmental periods, we show that such activity is also required at much earlier stages for spinal motoneurons to accurately execute their first major dorsal-ventral pathfinding decision. Blockade or slowing of rhythmic bursting activity also prevents the normal expression patterns of EphA4 and polysialic acid on NCAM, which may contribute to the pathfinding errors observed.