RESUMO
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. Deletion of ß- and γ-catenin from MN progenitors results in perinatal lethality and a severe reduction in phrenic MN bursting activity. In the absence of catenin signaling, phrenic MN topography is eroded, MN clustering is lost and phrenic axons and dendrites fail to grow appropriately. Despite the essential requirement for catenins in early phrenic MN development, they appear to be dispensable for phrenic MN maintenance, as catenin deletion from postmitotic MNs does not impact phrenic MN topography or function. Our data reveal a fundamental role for catenins in PMC development and suggest that distinct mechanisms are likely to control PMC maintenance.
Assuntos
Cateninas , Neurônios Motores , Gravidez , Feminino , Humanos , Neurônios Motores/fisiologia , Diafragma/inervação , Axônios , Transdução de SinaisRESUMO
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. Deletion of ß - and γ -catenin from MN progenitors results in perinatal lethality and a severe reduction in phrenic MN bursting activity. In the absence of catenin signaling, phrenic MN topography is eroded, MN clustering is lost and phrenic axons and dendrites fail to grow appropriately. Despite the essential requirement for catenins in early phrenic MN development, they appear to be dispensable for phrenic MN maintenance, as catenin deletion from postmitotic MNs does not impact phrenic MN topography or function. Our data reveal a fundamental role for catenins in PMC development and suggest that distinct mechanisms are likely to control PMC maintenance.
RESUMO
Breathing, and the motor circuits that control it, is essential for life. At the core of respiratory circuits are Dbx1-derived interneurons, which generate the rhythm and pattern of breathing, and phrenic motor neurons (MNs), which provide the final motor output that drives diaphragm muscle contractions during inspiration. Despite their critical function, the principles that dictate how respiratory circuits assemble are unknown. Here, we show that coordinated activity of a type I cadherin (N-cadherin) and type II cadherins (Cadherin-6, -9, and -10) is required in both MNs and Dbx1-derived neurons to generate robust respiratory motor output. Both MN- and Dbx1-specific cadherin inactivation in mice during a critical developmental window results in perinatal lethality due to respiratory failure and a striking reduction in phrenic MN bursting activity. This combinatorial cadherin code is required to establish phrenic MN cell body and dendritic topography; surprisingly, however, cell body position appears to be dispensable for the targeting of phrenic MNs by descending respiratory inputs. Our findings demonstrate that type I and II cadherins function cooperatively throughout the respiratory circuit to generate a robust breathing output and reveal novel strategies that drive the assembly of motor circuits.
The neural circuits which control breathing are established in the womb, ready to switch on with the first gulp of air. Defects in the way that this network is assembled can result in conditions such as sudden infant death syndrome. This process, however, remains poorly understood; in particular, it is still unclear exactly how the two main types of nerve cells which form respiratory circuits start to 'talk' to each other. Known as Dbx1-derived interneurons and phrenic motor neurons, these cell populations reside in different parts of the body and perform distinct roles. The interneurons, which are present in the brainstem, act as a pacemaker to set the rhythm of respiration; the motor neurons reside in the spinal cord, connecting the interneurons with the muscles which allow the lungs to fill with air. Vagnozzi et al. aimed to identify how phrenic motor neurons connect to and relay signals from other neurons involved in breathing to the diaphragm muscle. To do so, the team focused on cadherins, a group of proteins which allow cells to attach to one another. Studded through the membrane, these molecules are also often involved in forming connections from one cell to another that allow them to communicate. Newborn mice in which phrenic motor neurons lacked a specific combination of cadherins experienced respiratory failure, showing that these proteins were needed for breathing circuits to develop normally. Electrical activity recorded from these cells showed that phrenic motor neurons lacking cadherins could not receive the signals required to activate the breathing muscles. Microscopy imaging also revealed that the loss of cadherins shifted the position of the phrenic motor neurons within the spinal cord; however, this change did not seem to affect the connections these cells could establish. The ability to breathe is compromised in many incurable human diseases such as muscular dystrophies and amyotrophic lateral sclerosis. It may be possible to alleviate some of these symptoms by integrating phrenic motor neurons created in the laboratory into existing circuits. Studies which aim to decipher how the respiratory network is established, such as the one conducted by Vagnozzi et al., are essential in this effort.
Assuntos
Neurônios Motores , Respiração , Animais , Camundongos , Neurônios Motores/fisiologia , Interneurônios/fisiologia , Taxa Respiratória , Caderinas , Nervo Frênico , Proteínas de Homeodomínio/metabolismoRESUMO
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.
Assuntos
Genes Homeobox , Neurônios Motores/fisiologia , Nervo Frênico/fisiologia , Respiração/genética , Transcrição Gênica , Animais , CamundongosRESUMO
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. This article will review some of these contributions, highlighting several areas including the acquisition of motoneuron subtype identity and target selection, as well as the role of spontaneous rhythmic activity in circuit development.
Assuntos
Embrião de Galinha , Neurônios Motores/fisiologia , Neurônios/fisiologia , Codorniz/embriologia , Animais , Padronização Corporal , Galinhas , Biologia do Desenvolvimento , Extremidades , Camundongos , Músculos/embriologia , Músculos/inervação , PesquisaRESUMO
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. Growth cone contact with +/+ mouse myotubes resulted in immediate adhesive contacts and the rapid downregulation of growth cone motility. When contacting NCAM-/- myotubes, growth cones touched and retracted/collapsed multiple times and failed to form stable contacts, even after 10 h. Exogenous expression in myotubes of either the 180 or 140 isoform, but not the 120 kDa isoform, rescued the rapid formation of stable contacts, the accumulation of presynaptic and postsynaptic molecules, and functional transmission. When NCAM was absent only in motoneurons, growth cones did not retract upon myotube contact, but, since their motility was not downregulated, they grew off the ends of the myotubes, failing to form synapses. The agrin receptor Lrp4 was strongly downregulated in NCAM-negative myotubes, and motoneuron growth cones did not make stable contacts with Lrp4-negative myotubes. These studies have identified novel roles for presynaptic and postsynaptic NCAM in mediating early cell-cell interactions required for synapse formation.SIGNIFICANCE STATEMENT Although many molecular signals needed to form the functionally effective neuromuscular synapses required for normal movement have been described, the earliest signals that let motoneuron growth cones make stable adhesive contacts with myotubes and cease motility are not well understood. Using dynamic imaging of motoneuron-myotube cocultures, we show that NCAM is required on both the growth cone and myotube and that different NCAM isoforms mediate initial adhesion and the downregulation of growth cone motility. The agrin receptor Lrp4 was also essential for initial adhesive contacts and was downregulated on NCAM-/- myotubes. Our identification of novel roles for NCAM and Lrp4 and possible interactions between them in transforming motile growth cones into stable contacts opens interesting new avenues for exploration.
Assuntos
Cones de Crescimento/metabolismo , Neurônios Motores/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Neurogênese/fisiologia , Sinapses/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Junção Neuromuscular/crescimento & desenvolvimento , Junção Neuromuscular/metabolismo , Isoformas de ProteínasRESUMO
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.
Assuntos
Dióxido de Carbono/farmacologia , Expiração/efeitos dos fármacos , Inalação/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Centro Respiratório/efeitos dos fármacos , Taxa Respiratória/efeitos dos fármacos , Animais , Dióxido de Carbono/metabolismo , Expiração/fisiologia , Feminino , Nervo Hipoglosso/efeitos dos fármacos , Inalação/fisiologia , Masculino , Bulbo/citologia , Bulbo/efeitos dos fármacos , Bulbo/fisiologia , Camundongos , Neurônios/citologia , Neurônios/fisiologia , Optogenética/métodos , Nervo Frênico/efeitos dos fármacos , Picrotoxina/farmacologia , Prazosina/farmacologia , Propranolol/farmacologia , Centro Respiratório/citologia , Centro Respiratório/fisiologia , Taxa Respiratória/fisiologia , Raízes Nervosas Espinhais/efeitos dos fármacos , Estricnina/farmacologia , Substância P/farmacologiaRESUMO
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.
Assuntos
Vértebras Cervicais/fisiopatologia , Diafragma/inervação , Diafragma/fisiopatologia , Traumatismos da Medula Espinal/fisiopatologia , Animais , Animais Recém-Nascidos , Interneurônios/patologia , Luz , Vértebras Lombares/fisiopatologia , Camundongos , Neurônios Motores/patologia , Rede Nervosa/fisiopatologia , Paralisia/fisiopatologia , Nervo Frênico/fisiopatologia , Respiração , Transmissão Sináptica/fisiologia , Vértebras Torácicas/fisiopatologiaRESUMO
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. In embryos thus stimulated, motor axons correctly executed the binary dorsal-ventral pathfinding decision but failed to make the subsequent pool-specific decision to target to appropriate muscles. This observation, together with the previous demonstration that slowing the frequency by half perturbed dorsal-ventral but not pool-specific pathfinding, shows that modest changes in frequency differentially disrupt these two major pathfinding decisions. Thus, many drugs known to alter early rhythmic activity have the potential to impair normal motor circuit development, and given the conservation between mouse and avian spinal cords, our observations are likely relevant to mammals, where such studies would be difficult to carry out.
Assuntos
Potenciais de Ação/fisiologia , Axônios/fisiologia , Neurônios Motores/fisiologia , Optogenética/métodos , Potenciais de Ação/efeitos da radiação , Animais , Proteínas Aviárias/metabolismo , Embrião de Galinha , Eletromiografia , Luz , Modelos Neurológicos , Neurônios Motores/metabolismo , Contração Muscular/fisiologia , Contração Muscular/efeitos da radiação , Periodicidade , Músculo Quadríceps/embriologia , Músculo Quadríceps/fisiologia , Rodopsina/metabolismo , Nervos Espinhais/embriologia , Nervos Espinhais/fisiologiaRESUMO
A novel function for the neural cell adhesion molecule (NCAM) was identified in ephrinA/EphA-mediated repulsion as an important regulatory mechanism for development of GABAergic inhibitory synaptic connections in mouse prefrontal cortex. Deletion of NCAM, EphA3, or ephrinA2/3/5 in null mutant mice increased the numbers and size of perisomatic synapses between GABAergic basket interneurons and pyramidal cells in the developing cingulate cortex (layers II/III). A functional consequence of NCAM loss was increased amplitudes and faster kinetics of miniature inhibitory postsynaptic currents in NCAM null cingulate cortex. NCAM and EphA3 formed a molecular complex and colocalized with the inhibitory presynaptic marker vesicular GABA transporter (VGAT) in perisomatic puncta and neuropil in the cingulate cortex. EphrinA5 treatment promoted axon remodeling of enhanced green fluorescent protein-labeled basket interneurons in cortical slice cultures and induced growth cone collapse in wild-type but not NCAM null mutant neurons. NCAM modified with polysialic acid (PSA) was required to promote ephrinA5-induced axon remodeling of basket interneurons in cortical slices, likely by providing a permissive environment for ephrinA5/EphA3 signaling. These results reveal a new mechanism in which NCAM and ephrinAs/EphA3 coordinate to constrain GABAergic interneuronal arborization and perisomatic innervation, potentially contributing to excitatory/inhibitory balance in prefrontal cortical circuitry.
Assuntos
Efrinas/metabolismo , Neurônios GABAérgicos/fisiologia , Interneurônios/fisiologia , Moléculas de Adesão de Célula Nervosa/metabolismo , Plasticidade Neuronal/fisiologia , Córtex Pré-Frontal/fisiologia , Sinapses/fisiologia , Animais , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Ácido gama-Aminobutírico/metabolismoRESUMO
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. Here, we show that much less FM1-43 dye is lost per stimulus with 100 Hz stimulation than with 10 Hz stimulation even when the same number of vesicles undergo exocytosis. Electrophysiological data using folimycin show this lesser amount of dye loss is caused in part by the rapid reuse of vesicles. We showed previously that a myosin light chain kinase (MLCK)-myosin II pathway was required for effective transmission at 100 Hz. Here, we confirm the activation of MLCK, based on increased nerve terminal phospho-MLC immunostaining, with 100 Hz but not with 10 Hz stimulation. We further demonstrate that activation of MLCK, by increased extracellular Ca(2+), by PKC (protein kinase C) activation, or by a MLCK agonist peptide, reduces the amount of dye lost even with 10 Hz stimulation. MLCK activation at 10 Hz also resulted in more vesicles being rapidly reused. Thus, MLCK activation by 100 Hz stimulation switches the mechanism of vesicle cycling to a rapid-reuse mode and is required to sustain effective transmission in adult mouse NMJs.
Assuntos
Endocitose/fisiologia , Exocitose/fisiologia , Junção Neuromuscular/fisiologia , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/fisiologia , Análise de Variância , Animais , Cálcio/metabolismo , Eletrofisiologia , Imuno-Histoquímica , Camundongos , Neurônios Motores/fisiologia , Quinase de Cadeia Leve de Miosina/metabolismo , Proteína Quinase C/metabolismoRESUMO
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. Depleting but not abolishing the protein in motor neuronal progenitors causes an SMA-like phenotype. Neuromuscular weakness in the model mice is accompanied by peripheral as well as central synaptic defects, electrophysiological abnormalities of the neuromuscular junctions, muscle atrophy, and motor neuron degeneration. However, the disease phenotype is more modest than that observed in mice expressing ubiquitously low levels of the SMN protein, and both symptoms as well as early electrophysiological abnormalities that are readily apparent in neonates were attenuated in an age-dependent manner. We conclude that selective knock-down of SMN in motor neurons is sufficient but may not be necessary to cause a disease phenotype and that targeting these cells will be a requirement of any effective therapeutic strategy. This realization is tempered by the relatively mild SMA phenotype in our model mice, one explanation for which is the presence of normal SMN levels in non-neuronal tissue that serves to modulate disease severity.
Assuntos
Neurônios Motores/metabolismo , Neurônios Motores/patologia , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Células-Tronco/metabolismo , Fatores Etários , Análise de Variância , Animais , Animais Recém-Nascidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Comportamento Animal , Contagem de Células/métodos , Colina O-Acetiltransferase/metabolismo , Modelos Animais de Doenças , Eletromiografia/métodos , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Contração Isométrica/fisiologia , Estimativa de Kaplan-Meier , Proteínas Luminescentes/genética , Potenciais da Membrana/genética , Camundongos , Camundongos Transgênicos , Potenciais Pós-Sinápticos em Miniatura/genética , Atividade Motora/genética , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/mortalidade , Mutação/genética , Degeneração Neural/genética , Proteínas do Tecido Nervoso/metabolismo , Junção Neuromuscular/patologia , Fator de Transcrição 2 de Oligodendrócitos , Técnicas de Patch-Clamp , Receptores Colinérgicos/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo , Sinapses/patologia , Sinapses/fisiologia , Transmissão Sináptica/genéticaRESUMO
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. To distinguish whether the pathfinding errors were caused by perturbation of the normal frequency of bursting activity or interference with GABA(A) receptor signaling, chick embryos were chronically treated in ovo with picrotoxin to block GABA(A) receptors, while light activation by channelrhodopsin-2 was used to restore bursting activity to the control frequency. The restoration of normal patterns of neural activity in the presence of picrotoxin prevented the D-V pathfinding errors in the limb and maintained the normal expression levels of EphA4, EphB1, and polysialic acid on neural cell adhesion molecule, three molecules previously shown to be necessary for this pathfinding choice. These observations demonstrate that developing spinal motor circuits are highly sensitive to the precise frequency and pattern of spontaneous activity, and that any drugs that alter this activity could result in developmental defects.
Assuntos
Neurônios Motores/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de GABA-A/metabolismo , Rodopsina/metabolismo , Medula Espinal/metabolismo , Potenciais de Ação/fisiologia , Análise de Variância , Animais , Embrião de Galinha , Antagonistas GABAérgicos/farmacologia , Imuno-Histoquímica , Neurônios Motores/efeitos dos fármacos , Picrotoxina/farmacologia , Medula Espinal/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
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. We used enhanced yellow fluorescent protein under control of the Pet-1 enhancer/promoter region (ePet-EYFP), a specific marker of early differentiated serotonergic neurons, to study their migration via immunohistology and time-lapse imaging of living slice cultures. As early as E10.0, ePet-EYFP-expressing neurons were axonless, radially oriented, and spanned the entire neuroepithelium. The soma translocated within the pial process toward the pial surface and could also translocate through its neurites, which sprouted from the pial process. The dynamin inhibitor dynasore significantly reduced translocation velocity, while the nonmuscle myosin II inhibitor blebbistatin and the kinesin inhibitor AMP-PNP had no significant effect. Here we show for the first time that serotonergic neurons migrate by somal translocation mediated, in part, by dynamin.
Assuntos
Movimento Celular/fisiologia , Dinaminas/metabolismo , Epitélio/fisiologia , Matriz Extracelular/fisiologia , Neurônios/fisiologia , Serotonina/metabolismo , Fatores Etários , Animais , Proteínas de Bactérias/genética , Encéfalo/citologia , Inibidores Enzimáticos/farmacologia , Epitélio/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Hidrazonas/farmacologia , Técnicas In Vitro , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Gravidez , Regiões Promotoras Genéticas/genética , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Proteínas Proto-Oncogênicas/genéticaRESUMO
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. Some precursor cells in the ependymal layer displayed similar transients. T-type Ca(2+) channels and a persistent Na(+) current were essential to initiate spontaneous bursts and associated transients. However, subsequent propagation of activity throughout the cord resulted from network-driven chemical transmission mediated presynaptically by Ca(2+) entry through N-type Ca(2+) channels and postsynaptically by acetylcholine acting on nicotinic receptors. The increased [Ca(2+)](i) during transients depended primarily on L-type and T-type channels with a modest contribution from TRP (transient receptor potential) channels and ryanodine-sensitive internal stores. Significantly, the drugs used previously to produce pathfinding errors altered transient frequency but not duration or amplitude. These observations imply that different transient frequencies may differentially modulate motoneuron pathfinding. However, the duration of the Ca(2+) transients differed significantly between pools, potentially enabling additional distinct pool-specific downstream signaling. Many early events in spinal motor circuit formation are thus potentially sensitive to the rhythmic Ca(2+) transients we have characterized and to any drugs that perturb them.
Assuntos
Cálcio/metabolismo , Neurônios Motores/metabolismo , Periodicidade , Terminações Pré-Sinápticas/fisiologia , Receptores de Neurotransmissores/fisiologia , Potenciais de Ação/fisiologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Movimento Celular/fisiologia , Embrião de Galinha , Estimulação Elétrica/métodos , Células-Tronco Embrionárias/fisiologia , Estrenos/farmacologia , Técnicas In Vitro , Neurotransmissores/farmacologia , Compostos Orgânicos/metabolismo , Técnicas de Patch-Clamp/métodos , Inibidores de Fosfodiesterase/farmacologia , Pirrolidinonas/farmacologia , Receptores de Neurotransmissores/agonistas , Receptores de Neurotransmissores/antagonistas & inibidores , Medula Espinal/citologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
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. We show that the earliest structural defects appear distally and involve the neuromuscular synapse. Insufficient SMN protein arrests the post-natal development of the neuromuscular junction (NMJ), impairing the maturation of acetylcholine receptor (AChR) clusters into 'pretzels'. Pre-synaptic defects include poor terminal arborization and intermediate filament aggregates which may serve as a useful biomarker of the disease. These defects are reflected in functional deficits at the NMJ characterized by intermittent neurotransmission failures. We suggest that SMA might best be described as a NMJ synaptopathy and that one promising means of treating it could involve maintaining function at the NMJ.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Junção Neuromuscular/metabolismo , Junção Neuromuscular/fisiopatologia , Proteínas de Ligação a RNA/metabolismo , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Modelos Animais de Doenças , Feminino , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Neurônios Motores/química , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologia , Proteínas do Tecido Nervoso/genética , Junção Neuromuscular/genética , Junção Neuromuscular/patologia , Proteínas de Ligação a RNA/genética , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Proteínas do Complexo SMN , Proteína 2 de Sobrevivência do Neurônio Motor , Transmissão SinápticaRESUMO
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. A modest decrease in episode frequency resulted in dorsal-ventral pathfinding errors by lumbar motoneurons, and in the downregulation of several molecules required to successfully execute this guidance decision. In contrast, increasing the episode frequency was without effect on dorsal-ventral pathfinding. However, it prevented the subsequent motoneuron pool specific fasciculation of axons and their targeting to appropriate muscles, resulting in marked segmental pathfinding errors. These observations emphasize the need to better evaluate how such early spontaneous electrical activity may influence the molecular and transcription factor pathways that have been shown to regulate the differentiation of motor and interneuron phenotypes and the formation of spinal cord circuits. The intracellular signaling pathways by which episode frequency affects motor axon pathfinding must now be elucidated and it will be important to more precisely characterize the patterns with which specific subsets of motor and inter-neurons are activated normally and under conditions that alter spinal circuit formation.
Assuntos
Axônios/fisiologia , Galinhas/fisiologia , Vias Eferentes/crescimento & desenvolvimento , Neurônios Motores/fisiologia , Medula Espinal/fisiologia , Animais , Movimento Celular , Embrião de Galinha , Vias Eferentes/citologia , Extremidades/inervação , Extremidades/fisiologia , Medula Espinal/citologiaRESUMO
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. After myotube contact the 140 and 180 isoforms are downregulated from the axon and selectively targeted to the presynaptic terminal. This coincided with the downregulation of vesicle cycling along the axon and the expression of the mature form (BFA insensitive, P/Q type Ca2+ channel mediated) of vesicle cycling at the terminal. The synaptic targeting of exogenously expressed 180 and 140 isoforms also occurred when chick motoneurons contacted +/+ mouse myotubes; however only the 180 but not the 140 isoform was targeted on contact with NCAM-/- myotubes. These observations indicate that postsynaptic NCAM is required for the synaptic targeting of presynaptic 140 NCAM but that the localization of presynaptic 180 NCAM occurs via a different mechanism.
Assuntos
Neurônios Motores/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Moléculas de Adesão de Célula Nervosa/metabolismo , Junção Neuromuscular/citologia , Junção Neuromuscular/embriologia , Vesículas Sinápticas/fisiologia , Animais , Canais de Cálcio Tipo N/fisiologia , Células Cultivadas , Embrião de Galinha , Chlorocebus aethiops , Colina O-Acetiltransferase/metabolismo , Proteínas de Homeodomínio/metabolismo , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Medula Espinal/citologia , Proteína 25 Associada a Sinaptossoma/metabolismo , Transfecção/métodosRESUMO
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. Light-activated G-protein-coupled receptors and ion channels, which can be genetically manipulated and targeted to neuronal circuits, have the greatest potential to fulfill these requirements.
Assuntos
Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Transmissão Sináptica/fisiologia , Visão Ocular/fisiologia , Animais , Humanos , Canais Iônicos/fisiologia , Estimulação Luminosa/métodos , Células Fotorreceptoras de Invertebrados/ultraestrutura , Células Fotorreceptoras de Vertebrados/ultraestrutura , Receptores Acoplados a Proteínas G/fisiologia , Retinoides/metabolismo , Rodopsina/metabolismoRESUMO
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. Moderate speeding up of activity is shown here not to affect these molecules or D-V pathfinding but to strongly perturb the anteroposterior (A-P) pathfinding process by which motoneurons fasciculate into pool-specific fascicles at the limb base and then selectively grow to muscle targets. Resumption of normal frequency allowed axons to correct the A-P pathfinding errors by altering their trajectories distally, indicating the dynamic nature of this process and its continued sensitivity to patterned activity.