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Advanced Healthcare Materials Jan 2016The brain is one of the most important and complex organs in the human body. Although various neural network models have been proposed for in vitro 3D neuronal networks,...
The brain is one of the most important and complex organs in the human body. Although various neural network models have been proposed for in vitro 3D neuronal networks, it has been difficult to mimic functional and structural complexity of the in vitro neural circuit. Here, a microfluidic model of a simplified 3D neural circuit is reported. First, the microfluidic device is filled with Matrigel and continuous flow is delivered across the device during gelation. The fluidic flow aligns the extracellular matrix (ECM) components along the flow direction. Following the alignment of ECM fibers, neurites of primary rat cortical neurons are grown into the Matrigel at the average speed of 250 μm d(-1) and form axon bundles approximately 1500 μm in length at 6 days in vitro (DIV). Additionally, neural networks are developed from presynaptic to postsynaptic neurons at 14 DIV. The establishment of aligned 3D neural circuits is confirmed with the immunostaining of PSD-95 and synaptophysin and the observation of calcium signal transmission.
Topics: Animals; Collagen; Cross-Linking Reagents; Drug Combinations; Extracellular Matrix; Hydrogel, Polyethylene Glycol Dimethacrylate; Laminin; Microfluidics; Neurites; Neurons; Proteoglycans; Rats, Sprague-Dawley; Tissue Engineering
PubMed: 26332914
DOI: 10.1002/adhm.201500397 -
The Journal of Comparative Neurology May 2018Prior to forming and refining synaptic connections, axons of projection neurons navigate long distances to their targets. While much is known about guidance cues for...
Prior to forming and refining synaptic connections, axons of projection neurons navigate long distances to their targets. While much is known about guidance cues for axon navigation through intermediate choice points, whether and how axons are organized within tracts is less clear. Here we analyze the organization of retinal ganglion cell (RGC) axons in the developing mouse retinogeniculate pathway. RGC axons are organized by both eye-specificity and topography in the optic nerve and tract: ipsilateral RGC axons are segregated from contralateral axons and are offset laterally in the tract relative to contralateral axon topographic position. To identify potential cell-autonomous factors contributing to the segregation of ipsilateral and contralateral RGC axons in the visual pathway, we assessed their fasciculation behavior in a retinal explant assay. Ipsilateral RGC neurites self-fasciculate more than contralateral neurites in vitro and maintain this difference in the presence of extrinsic chiasm cues. To further probe the role of axon self-association in circuit formation in vivo, we examined RGC axon organization and fasciculation in an EphB1 mutant, in which a subset of ipsilateral RGC axons aberrantly crosses the midline but targets the ipsilateral zone in the dorsal lateral geniculate nucleus on the opposite side. Aberrantly crossing axons retain their association with ipsilateral axons in the contralateral tract, indicating that cohort-specific axon affinity is maintained independently of guidance signals present at the midline. Our results provide a comprehensive assessment of RGC axon organization in the retinogeniculate pathway and suggest that axon self-association contributes to pre-target axon organization.
Topics: Amino Acids; Animals; Animals, Newborn; Axons; Embryo, Mammalian; Eye; Fasciculation; Functional Laterality; In Vitro Techniques; Intermediate Filaments; Luminescent Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Optic Nerve; Receptor, EphB1; Retinal Ganglion Cells; Serotonin Plasma Membrane Transport Proteins; Visual Pathways
PubMed: 29322522
DOI: 10.1002/cne.24392 -
The Journal of Neuroscience : the... Aug 2023Semaphorins and Plexins form ligand/receptor pairs that are crucial for a wide range of developmental processes from cell proliferation to axon guidance. The ability of...
Semaphorins and Plexins form ligand/receptor pairs that are crucial for a wide range of developmental processes from cell proliferation to axon guidance. The ability of semaphorins to act both as signaling receptors and ligands yields a multitude of responses. Here, we describe a novel role for Semaphorin-6D (Sema6D) and Plexin-A1 in the positioning and targeting of retinogeniculate axons. In or mutant mice of either sex, the optic tract courses through, rather than along, the border of the dorsal lateral geniculate nucleus (dLGN), and some retinal axons ectopically arborize adjacent and lateral to the optic tract rather than defasciculating and entering the target region. We find that Sema6D and Plexin-A1 act together in a dose-dependent manner, as the number of the ectopic retinal projections is altered in proportion to the level of Sema6D or Plexin-A1 expression. Moreover, using retinal electroporation of Sema6D or Plexin-A1 shRNA, we show that Sema6D and Plexin-A1 are both required in retinal ganglion cells for axon positioning and targeting. Strikingly, nonelectroporated retinal ganglion cell axons also mistarget in the tract region, indicating that Sema6D and Plexin-A1 can act non-cell-autonomously, potentially through axon-axon interactions. These data provide novel evidence for a dose-dependent and non-cell-autonomous role for Sema6D and Plexin-A1 in retinal axon organization in the optic tract and dLGN. Before innervating their central brain targets, retinal ganglion cell axons fasciculate in the optic tract and then branch and arborize in their target areas. Upon deletion of the guidance molecules Plexin-A1 or Semaphorin-6D, the optic tract becomes disorganized near and extends within the dorsal lateral geniculate nucleus. In addition, some retinal axons form ectopic aggregates within the defasciculated tract. Sema6D and Plexin-A1 act together as a receptor-ligand pair in a dose-dependent manner, and non-cell-autonomously, to produce this developmental aberration. Such a phenotype highlights an underappreciated role for axon guidance molecules in tract cohesion and appropriate defasciculation near, and arborization within, targets.
Topics: Animals; Mice; Axons; Ligands; Retinal Ganglion Cells; Semaphorins
PubMed: 37344233
DOI: 10.1523/JNEUROSCI.0072-22.2023 -
Scientific Reports Nov 2014The accuracy of axonal pathfinding and the formation of functional neural circuitry are crucial for an organism to process, store, and retrieve information from internal...
The accuracy of axonal pathfinding and the formation of functional neural circuitry are crucial for an organism to process, store, and retrieve information from internal networks as well as from the environment. Aberrations in axonal migration is believed to lead to loop formation and self-fasciculation, which can lead to highly dysfunctional neural circuitry and therefore self-avoidance of axons is proposed to be the regulatory mechanism for control of synaptogenesis. Here, we report the application of a newly developed non-contact optical method using a weakly-focused, near infrared laser beam for highly efficient axonal guidance, and demonstrate the formation of axonal loops in cortical neurons, which demonstrate that cortical neurons can self-fasciculate in contrast to self-avoidance. The ability of light for axonal nano-loop formation opens up new avenues for the construction of complex neural circuitry, and non-invasive guidance of neurons at long working distances for restoration of impaired neural connections and functions.
Topics: Animals; Axons; Cerebral Cortex; Embryo, Mammalian; Fasciculation; Infrared Rays; Kinetics; Lasers; Nerve Net; Neurogenesis; Photic Stimulation; Photons; Primary Cell Culture; Rats
PubMed: 25376602
DOI: 10.1038/srep06902 -
FEBS Open Bio Jan 2018Fasciculation and elongation zeta-1 (FEZ1) protein is involved in axon outgrowth and is highly expressed in the brain. It has multiple interaction partners, with...
Fasciculation and elongation zeta-1 (FEZ1) protein is involved in axon outgrowth and is highly expressed in the brain. It has multiple interaction partners, with functions varying from the regulation of neuronal development and intracellular transport mechanisms to transcription regulation. One of its interactors is retinoic acid receptor (RAR), which is activated by retinoic acid and controls many target genes and physiological process. Based on previous evidence suggesting a possible nuclear role for FEZ1, we wanted to deepen our understanding of this function by addressing the FEZ1-RAR interaction. We performed binding experiments and assessed the interface of interaction between both proteins. We found that FEZ1-RAR interacted with a similar magnitude as RAR to its responsive element DR5 and that the interaction occurred in the coiled-coil region of FEZ1 and in the ligand-binding domain of RAR. Furthermore, cellular experiments were performed in order to confirm the interaction and screen for induced target genes from an 86-gene panel. The analysis of gene expression showed that only in the presence of retinoic acid did FEZ1 induce gene expression. This finding is consistent with data from the literature showing the gene functionally involved in development and acute myeloid leukemia, as is FEZ1.
PubMed: 29321952
DOI: 10.1002/2211-5463.12338 -
Cell Reports Aug 2018Length-dependent axonopathy of the corticospinal tract causes lower limb spasticity and is characteristic of several neurological disorders, including hereditary spastic...
Length-dependent axonopathy of the corticospinal tract causes lower limb spasticity and is characteristic of several neurological disorders, including hereditary spastic paraplegia (HSP) and amyotrophic lateral sclerosis. Mutations in Trk-fused gene (TFG) have been implicated in both diseases, but the pathomechanisms by which these alterations cause neuropathy remain unclear. Here, we biochemically and genetically define the impact of a mutation within the TFG coiled-coil domain, which underlies early-onset forms of HSP. We find that the TFG (p.R106C) mutation alters compaction of TFG ring complexes, which play a critical role in the export of cargoes from the endoplasmic reticulum (ER). Using CRISPR-mediated genome editing, we engineered human stem cells that express the mutant form of TFG at endogenous levels and identified specific defects in secretion from the ER and axon fasciculation following neuronal differentiation. Together, our data highlight a key role for TFG-mediated protein transport in the pathogenesis of HSP.
Topics: Axon Fasciculation; Axons; Base Sequence; Humans; Mutation; Neurons; Protein Transport; Proteins; Spastic Paraplegia, Hereditary
PubMed: 30157421
DOI: 10.1016/j.celrep.2018.07.081 -
Journal of Tissue Engineering and... Feb 2022Three dimensional (3D) in vitro neuronal cultures can better reproduce physiologically relevant phenotypes compared to 2D-cultures, because in vivo neurons reside in a...
Three dimensional (3D) in vitro neuronal cultures can better reproduce physiologically relevant phenotypes compared to 2D-cultures, because in vivo neurons reside in a 3D microenvironment. Interest in neuronal 3D cultures is emerging, with special attention to the mechanical forces that regulate axon elongation and sprouting in three dimensions. Type I collagen (Col-I) is a native substrate since it is present in the extracellular matrix and hence emulates an in vivo environment to study axon growth. The impact of its mechanical properties needs to be further investigated. Here, we generated Col-I 3D matrices of different mechanical stiffness and evaluated axon growth in three dimensions. Superior cervical ganglion (SCG) explants from neonatal rats were cultured in soft and stiff Col-I 3D matrices and neurite outgrowth was assessed by measuring: maximum neuritic extent; neuritic halo area and fasciculation. Axonal cytoskeletal proteins were examined. Axon elongation in stiff Col-I 3D matrices was reduced (31%) following 24 h in culture compared to soft matrices. In stiff matrices, neurites fasciculated and formed less dense halos. Consistently, almost no F-actin rich growth cones were recognized, and F-actin staining was strongly reduced in the axonal compartment. This study shows that stiffness negatively affects 3D neurite outgrowth and adds insights on the cytoskeletal responses upon mechanic interactions of axons with a 3D environment. Our data will serve to facilitate the development of model systems that are mechanically well-behaved but still mimic key physiologic properties observed in vivo.
Topics: Actins; Animals; Axons; Cells, Cultured; Collagen Type I; Extracellular Matrix; Growth Cones; Neurites; Rats
PubMed: 34816618
DOI: 10.1002/term.3269 -
Development Genes and Evolution Dec 2023Johnston's organ (Jo) acts as an antennal wind-sensitive and/or auditory organ across a spectrum of insect species and its axons universally project to the brain. In the...
Johnston's organ (Jo) acts as an antennal wind-sensitive and/or auditory organ across a spectrum of insect species and its axons universally project to the brain. In the locust, this pathway is already present at mid-embryogenesis but the process of fasciculation involved in its construction has not been investigated. Terminal projections into the fine neuropilar organization of the brain also remain unresolved, information essential not only for understanding the neural circuitry mediating Jo-mediated behavior but also for providing comparative data offering insights into its evolution. In our study here, we employ neuron-specific, axon-specific, and epithelial domain labels to show that the pathway to the brain of the locust is built in a stepwise manner during early embryogenesis as processes from Jo cell clusters in the pedicel fasciculate first with one another, and then with the two tracts constituting the pioneer axon scaffold of the antenna. A comparison of fasciculation patterns confirms that projections from cell clusters of Jo stereotypically associate with only one axon tract according to their location in the pedicellar epithelium, consistent with a topographic plan. At the molecular level, all neuronal elements of the Jo pathway to the brain express the lipocalin Lazarillo, a cell surface epitope that regulates axogenesis in the primary axon scaffold itself, and putatively during fasciculation of the Jo projections to the brain. Central projections from Jo first contact the primary axon scaffold of the deutocerebral brain at mid-embryogenesis, and in the adult traverse mechanosensory/motor neuropils similar to those in Drosophila. These axons then terminate among protocerebral commissures containing premotor interneurons known to regulate flight behavior.
Topics: Animals; Grasshoppers; Fasciculation; Neurons; Brain; Drosophila
PubMed: 37695323
DOI: 10.1007/s00427-023-00710-0 -
Neuron Aug 2014To explore the link between bioenergetics and motor neuron degeneration, we used a computational model in which detailed morphology and ion conductance are paired with...
To explore the link between bioenergetics and motor neuron degeneration, we used a computational model in which detailed morphology and ion conductance are paired with intracellular ATP production and consumption. We found that reduced ATP availability increases the metabolic cost of a single action potential and disrupts K+/Na+ homeostasis, resulting in a chronic depolarization. The magnitude of the ATP shortage at which this ionic instability occurs depends on the morphology and intrinsic conductance characteristic of the neuron. If ATP shortage is confined to the distal part of the axon, the ensuing local ionic instability eventually spreads to the whole neuron and involves fasciculation-like spiking events. A shortage of ATP also causes a rise in intracellular calcium. Our modeling work supports the notion that mitochondrial dysfunction can account for salient features of the paralytic disorder amyotrophic lateral sclerosis, including motor neuron hyperexcitability, fasciculation, and differential vulnerability of motor neuron subpopulations.
Topics: Action Potentials; Adenosine Triphosphate; Calcium; Energy Metabolism; Homeostasis; Models, Neurological; Motor Neurons; Nerve Degeneration; Potassium; Sodium
PubMed: 25088365
DOI: 10.1016/j.neuron.2014.07.001 -
Methods in Molecular Biology (Clifton,... 2017How are precise connectivity to peripheral targets and corresponding sensory-motor networks established during developmental innervation of the vertebrate extremities?...
How are precise connectivity to peripheral targets and corresponding sensory-motor networks established during developmental innervation of the vertebrate extremities? The formation of functional sensory-motor circuits requires highly appropriate temporal and spatial regulation of axon growth which is achieved through the combination of different molecular mechanisms such as communication between heterotypic fiber systems, axon-environment, or axon-glia interactions that ensure proper fasciculation and accurate pathfinding to distal targets. Family members of the class 3 semaphorins and their cognate receptors, the neuropilins, were shown to govern various events during wiring of central and peripheral circuits, with mice lacking Sema3-Npn signaling showing deficits in timing of growth, selective fasciculation, guidance fidelity, and coupling of sensory axon growth to motor axons at developmental time points. Given the accuracy with which these processes have to interact in a stepwise manner, deficiency of the smallest cog in the wheel may impact severely on the faithful establishment and functionality of peripheral circuitries, ultimately leading to behavioral impairments or even cause the death of the animal. Reliable quantitative analyses of sensory-motor fasciculation, extension, and guidance of axons to their cognate target muscles and the skin during development, but also assessment of physiological and behavioral consequences at adult age, are therefore a necessity to extend our understanding of the molecular mechanisms of peripheral circuit formation. In this chapter we provide a detailed methodology to characterize class 3 semaphorin-mediated effects on peripheral sensory and motor axon pathfinding and connectivity during embryonic development.
Topics: Animals; Axon Guidance; Axons; Embryonic Development; Female; Mice; Pregnancy; Semaphorins
PubMed: 27787870
DOI: 10.1007/978-1-4939-6448-2_32