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ACS Biomaterials Science & Engineering Feb 2022Relative to two-dimensional (2D) culture, three-dimensional (3D) culture of primary neurons has yielded increasingly physiological responses from cells. Electrospun...
Relative to two-dimensional (2D) culture, three-dimensional (3D) culture of primary neurons has yielded increasingly physiological responses from cells. Electrospun nanofiber scaffolds are frequently used as a 3D biomaterial support for primary neurons in neural tissue engineering, while hydrophobic surfaces typically induce aggregation of cells. Poly-l-lactic acid (PLLA) was electrospun as aligned PLLA nanofiber scaffolds to generate a structure with both qualities. Primary cortical neurons from E18 Sprague-Dawley rats cultured on aligned PLLA nanofibers generated 3D clusters of cells that extended highly aligned, fasciculated neurite bundles within 10 days. These clusters were viable for 28 days and responsive to AMPA and GABA. Relative to the 2D culture, the 3D cultures exhibited a more developed profile; mass spectrometry demonstrated an upregulation of proteins involved in cortical lamination, polarization, and axon fasciculation and a downregulation of immature neuronal markers. The use of artificial neural network inference suggests that the increased formation of synapses may drive the increase in development that is observed for the 3D cell clusters. This research suggests that aligned PLLA nanofibers may be highly useful for generating advanced 3D cell cultures for high-throughput systems.
Topics: Animals; Nanofibers; Neurons; Polyesters; Rats; Rats, Sprague-Dawley; Tissue Scaffolds
PubMed: 35084839
DOI: 10.1021/acsbiomaterials.1c01102 -
Scientific Reports Apr 2019Ceramide phosphoethanolamine (CPE), a major sphingolipid in invertebrates, is crucial for axonal ensheathment in Drosophila. Darkfield microscopy revealed that an...
Ceramide phosphoethanolamine (CPE), a major sphingolipid in invertebrates, is crucial for axonal ensheathment in Drosophila. Darkfield microscopy revealed that an equimolar mixture of bovine buttermilk CPE (milk CPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (diC18:1 PC) tends to form tubules and helical ribbons, while pure milk CPE mainly exhibits amorphous aggregates and, at low frequency, straight needles. Negative staining electron microscopy indicated that helices and tubules were composed of multilayered 5-10 nm thick slab-like structures. Using different molecular species of PC and CPE, we demonstrated that the acyl chain length of CPE but not of PC is crucial for the formation of tubules and helices in equimolar mixtures. Incubation of the lipid suspensions at the respective phase transition temperature of CPE facilitated the formation of both tubules and helices, suggesting a dynamic lipid rearrangement during formation. Substituting diC18:1 PC with diC18:1 PE or diC18:1 PS failed to form tubules and helices. As hydrated galactosylceramide (GalCer), a major lipid in mammalian myelin, has been reported to spontaneously form tubules and helices, it is believed that the ensheathment of axons in mammals and Drosophila is based on similar physical processes with different lipids.
Topics: Animals; Axon Fasciculation; Drosophila; Galactosylceramides; Lipid Bilayers; Membranes; Molecular Conformation; Nervous System; Phase Transition; Phosphatidylcholines; Sphingomyelins
PubMed: 30967612
DOI: 10.1038/s41598-019-42247-1 -
The Journal of Neuroscience : the... Apr 2017Membrane excitability in the axonal growth cones of embryonic neurons influences axon growth. Voltage-gated K (Kv) channels are key factors in controlling membrane...
Membrane excitability in the axonal growth cones of embryonic neurons influences axon growth. Voltage-gated K (Kv) channels are key factors in controlling membrane excitability, but whether they regulate axon growth remains unclear. Here, we report that Kv3.4 is expressed in the axonal growth cones of embryonic spinal commissural neurons, motoneurons, dorsal root ganglion neurons, retinal ganglion cells, and callosal projection neurons during axon growth. Our (cultured dorsal spinal neurons of chick embryos) and (developing chick spinal commissural axons and rat callosal axons) findings demonstrate that knockdown of Kv3.4 by a specific shRNA impedes axon initiation, elongation, pathfinding, and fasciculation. In cultured dorsal spinal neurons, blockade of Kv3.4 by blood depressing substance II suppresses axon growth via an increase in the amplitude and frequency of Ca influx through T-type and L-type Ca channels. Electrophysiological results show that Kv3.4, the major Kv channel in the axonal growth cones of embryonic dorsal spinal neurons, is activated at more hyperpolarized potentials and inactivated more slowly than it is in postnatal and adult neurons. The opening of Kv3.4 channels effectively reduces growth cone membrane excitability, thereby limiting excessive Ca influx at subthreshold potentials or during Ca-dependent action potentials. Furthermore, excessive Ca influx induced by an optogenetic approach also inhibits axon growth. Our findings suggest that Kv3.4 reduces growth cone membrane excitability and maintains [Ca] at an optimal concentration for normal axon growth. Accumulating evidence supports the idea that impairments in axon growth contribute to many clinical disorders, such as autism spectrum disorders, corpus callosum agenesis, Joubert syndrome, Kallmann syndrome, and horizontal gaze palsy with progressive scoliosis. Membrane excitability in the growth cone, which is mainly controlled by voltage-gated Ca (Cav) and K (Kv) channels, modulates axon growth. The role of Cav channels during axon growth is well understood, but it is unclear whether Kv channels control axon outgrowth by regulating Ca influx. This report shows that Kv3.4, which is transiently expressed in the axonal growth cones of many types of embryonic neurons, acts to reduce excessive Ca influx through Cav channels and thus permits normal axon outgrowth.
Topics: Action Potentials; Animals; Axons; Calcium; Chick Embryo; Corpus Callosum; Electroporation; Ganglia, Spinal; Gene Knockdown Techniques; Growth Cones; Motor Neurons; Neurons; Potassium Channels, Voltage-Gated; Rats; Retinal Ganglion Cells
PubMed: 28320840
DOI: 10.1523/JNEUROSCI.1076-16.2017 -
Mechanisms of Development Aug 2018Contactin2 (Cntn2)/Transient Axonal Glycoprotein 1 (Tag1), a neural cell adhesion molecule, has established roles in neuronal migration and axon fasciculation in chick...
Contactin2 (Cntn2)/Transient Axonal Glycoprotein 1 (Tag1), a neural cell adhesion molecule, has established roles in neuronal migration and axon fasciculation in chick and mouse. In zebrafish, antisense morpholino-based studies have indicated roles for cntn2 in the migration of facial branchiomotor (FBM) neurons, the guidance of the axons of the nucleus of the medial longitudinal fascicle (nucMLF), and the outgrowth of Rohon-Beard (RB) central axons. To study functions of Cntn2 in later stages of neuronal development, we generated cntn2 mutant zebrafish using CRISPR-Cas9. Using a null mutant allele, we detected genetic interactions between cntn2 and the planar cell polarity gene vangl2, as shown previously with cntn2 morphants, demonstrating a function for cntn2 during FBM neuron migration in a sensitized background of reduced planar cell polarity signaling. In addition, maternal-zygotic (MZ) cntn2 mutant larvae exhibited aberrant touch responses and swimming, suggestive of defects in sensorimotor circuits, consistent with studies in mice. However, the nucMLF axon convergence, FBM neuron migration, and RB outgrowth defects seen in morphants were not seen in the mutants, and we show here that they are likely off-target effects of morpholinos. However, MLF axons exhibited local defasciculation in MZcntn2 mutants, consistent with a role for Cntn2 in axon fasciculation. These data demonstrate distinct roles for zebrafish cntn2 in neuronal migration and axon fasciculation, and in the function of sensorimotor circuits.
Topics: Animals; Axons; CRISPR-Cas Systems; Cell Adhesion; Cell Movement; Cell Polarity; Contactin 2; Gene Expression Regulation, Developmental; Humans; Mice; Morpholinos; Motor Neurons; Neurogenesis; Zebrafish; Zebrafish Proteins
PubMed: 29777776
DOI: 10.1016/j.mod.2018.05.005 -
Development (Cambridge, England) Jun 2020Thalamocortical axons (TCAs) cross several tissues on their journey to the cortex. Mechanisms must be in place along the route to ensure they connect with their targets...
Thalamocortical axons (TCAs) cross several tissues on their journey to the cortex. Mechanisms must be in place along the route to ensure they connect with their targets in an orderly fashion. The ventral telencephalon acts as an instructive tissue, but the importance of the diencephalon in TCA mapping is unknown. We report that disruption of diencephalic development by Pax6 deletion results in a thalamocortical projection containing mapping errors. We used conditional mutagenesis to test whether these errors are due to the disruption of pioneer projections from prethalamus to thalamus and found that, although this correlates with abnormal TCA fasciculation, it does not induce topographical errors. To test whether the thalamus contains navigational cues for TCAs, we used slice culture transplants and gene expression studies. We found the thalamic environment is instructive for TCA navigation and that the molecular cues netrin 1 and semaphorin 3a are likely to be involved. Our findings indicate that the correct topographic mapping of TCAs onto the cortex requires the order to be established from the earliest stages of their growth by molecular cues in the thalamus itself.
Topics: Animals; Axons; Diencephalon; Embryo, Mammalian; Gene Expression Regulation, Developmental; Homeodomain Proteins; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Knockout; Mutagenesis; Netrin-1; Organ Culture Techniques; PAX6 Transcription Factor; Semaphorin-3A; Thalamus
PubMed: 32541009
DOI: 10.1242/dev.184523 -
Gene Feb 2015ZFR is an ancient and highly conserved chromosome-associated protein from nematodes to mammals, embryologically expressed in most species, with the exception of the...
ZFR is an ancient and highly conserved chromosome-associated protein from nematodes to mammals, embryologically expressed in most species, with the exception of the nematode Caenorhabditis elegans. The ZFR encodes zinc and RNA binding protein, and in rat, the nuclear-cytoplasmic shuttling ZFR has been found with transport and translation-associated RNA granule-like structures in the somatodendritic compartments of hippocampal neurons. The majority of axons cross the midline before projecting to their contralateral synaptic target and this crossing decision is under tight control. Molecular factors contributing to these processes have been identified, although the mechanisms are not fully understood. In this study, we tested the role of ceZFR in axon guidance using ceZfr RNAi-treated animals to analyse axon midline crossing, axon fasciculation and cord commissures. In adult stages, RNAi-induced depletion of the ceZfr transcript leads to several phenotypes related to axon guidance. A midline crossing defect was observed in the ventral nerve cord (VNC) in axon type D, DD/VD motoneuron axons and axon type 1, interneuron axons. We further detected a dorsal nerve cord (DNC) axon fasciculation. Some ceZfr RNAi-treated animals revealed that cord commissures fail to reach their synaptic target. We provide evidence that ceZFR has a role in axon guidance. When Zfr was depleted by RNAi, the phenotypes are characterized by defects in axon midline crossing, axon defasciculation and cord commissures. Our results thus support the hypothesis that ZFR has essential roles during neurogenesis, and could support early steps of RNA transport and localization through RNA granule formation in the nucleus and/or to their nucleo-cytoplasmic shuttling.
Topics: Amino Acid Sequence; Animals; Axons; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cytoplasmic Granules; Gene Expression Regulation, Developmental; Interneurons; Molecular Sequence Data; Motor Neurons; Neurogenesis; RNA Interference; RNA Transport; RNA, Small Interfering; RNA-Binding Proteins; Zinc Fingers
PubMed: 25476027
DOI: 10.1016/j.gene.2014.11.063 -
Development (Cambridge, England) Aug 2020Fragile X mental retardation protein (FMRP) is an RNA-binding protein abundant in the nervous system. Functional loss of FMRP leads to sensory dysfunction and severe...
Fragile X mental retardation protein (FMRP) is an RNA-binding protein abundant in the nervous system. Functional loss of FMRP leads to sensory dysfunction and severe intellectual disabilities. In the auditory system, FMRP deficiency alters neuronal function and synaptic connectivity and results in perturbed processing of sound information. Nevertheless, roles of FMRP in embryonic development of the auditory hindbrain have not been identified. Here, we developed high-specificity approaches to genetically track and manipulate throughout development of the Atoh1 neuronal cell type, which is highly conserved in vertebrates, in the cochlear nucleus of chicken embryos. We identified distinct FMRP-containing granules in the growing axons of Atoh1 neurons and post-migrating NM cells. FMRP downregulation induced by CRISPR/Cas9 and shRNA techniques resulted in perturbed axonal pathfinding, delay in midline crossing, excess branching of neurites, and axonal targeting errors during the period of circuit development. Together, these results provide the first identification of FMRP localization and actions in developing axons of auditory neurons, and demonstrate the importance of investigating early embryonic alterations toward understanding the pathogenesis of neurodevelopmental disorders.
Topics: Animals; Auditory Pathways; Axons; Base Sequence; CRISPR-Cas Systems; Chick Embryo; Chickens; Dendrites; Fragile X Mental Retardation Protein; Neural Stem Cells; Presynaptic Terminals; RNA, Small Interfering; Rhombencephalon; Synapses; Time Factors
PubMed: 32747436
DOI: 10.1242/dev.188797 -
Frontiers in Neuroscience 2023The protein fasciculation and elongation zeta-1 (FEZ1) is involved in axon outgrowth but potentially interacts with various proteins with roles ranging from...
INTRODUCTION
The protein fasciculation and elongation zeta-1 (FEZ1) is involved in axon outgrowth but potentially interacts with various proteins with roles ranging from intracellular transport to transcription regulation. Gene association and other studies have identified as being directly, or indirectly, implicated in schizophrenia susceptibility. To explore potential roles in normal early human forebrain neurodevelopment, we mapped expression by region and cell type.
METHODS
All tissues were provided with maternal consent and ethical approval by the Human Developmental Biology Resource. RNAseq data were obtained from previously published sources. Thin paraffin sections from 8 to 21 post-conceptional weeks (PCW) samples were used for RNAScope hybridization and immunohistochemistry against mRNA and protein, and other marker proteins.
RESULTS
Tissue RNAseq revealed that is highly expressed in the human cerebral cortex between 7.5-17 PCW and single cell RNAseq at 17-18 PCW confirmed its expression in all neuroectoderm derived cells. The highest levels were found in more mature glutamatergic neurons, the lowest in GABAergic neurons and dividing progenitors. In the thalamus, single cell RNAseq similarly confirmed expression in multiple cell types. In cerebral cortex sections at 8-10 PCW, strong expression of mRNA and protein appeared confined to post-mitotic neurons, with low expression seen in progenitor zones. Protein expression was observed in some axon tracts by 16-19 PCW. However, in sub-cortical regions, was highly expressed in progenitor zones at early developmental stages, showing lower expression in post-mitotic cells.
DISCUSSION
FEZ1 has different expression patterns and potentially diverse functions in discrete forebrain regions during prenatal human development.
PubMed: 37746155
DOI: 10.3389/fnins.2023.1249973 -
Scientific Reports Oct 2017During nervous system development growing axons can interact with each other, for example by adhering together in order to produce bundles (fasciculation). How does such...
During nervous system development growing axons can interact with each other, for example by adhering together in order to produce bundles (fasciculation). How does such axon-axon interaction affect the resulting axonal trajectories, and what are the possible benefits of this process in terms of network function? In this paper we study these questions by adapting an existing computational model of the development of neurons in the Xenopus tadpole spinal cord to include interactions between axons. We demonstrate that even relatively weak attraction causes bundles to appear, while if axons weakly repulse each other their trajectories diverge such that they fill the available space. We show how fasciculation can help to ensure axons grow in the correct location for proper network formation when normal growth barriers contain gaps, and use a functional spiking model to show that fasciculation allows the network to generate reliable swimming behaviour even when overall synapse counts are artificially lowered. Although we study fasciculation in one particular organism, our approach to modelling axon growth is general and can be widely applied to study other nervous systems.
Topics: Animals; Axon Fasciculation; Larva; Models, Biological; Spinal Cord; Synapses; Xenopus laevis
PubMed: 29051550
DOI: 10.1038/s41598-017-13804-3 -
ELife Sep 2017Nervous system function relies on precise synaptic connections. A number of widely-conserved cell adhesion proteins are implicated in cell recognition between synaptic...
Nervous system function relies on precise synaptic connections. A number of widely-conserved cell adhesion proteins are implicated in cell recognition between synaptic partners, but how these proteins act as a group to specify a complex neural network is poorly understood. Taking advantage of known connectivity in , we identified and studied cell adhesion genes expressed in three interacting neurons in the mating circuits of the adult male. Two interacting pairs of cell surface proteins independently promote fasciculation between sensory neuron HOA and its postsynaptic target interneuron AVG: BAM-2/neurexin-related in HOA binds to CASY-1/calsyntenin in AVG; SAX-7/L1CAM in sensory neuron PHC binds to RIG-6/contactin in AVG. A third, basal pathway results in considerable HOA-AVG fasciculation and synapse formation in the absence of the other two. The features of this multiplexed mechanism help to explain how complex connectivity is encoded and robustly established during nervous system development.
Topics: Animals; Caenorhabditis elegans; Cell Adhesion; Connectome; Gene Expression Profiling; Male; Nerve Net; Neural Cell Adhesion Molecules; Neurons
PubMed: 28901288
DOI: 10.7554/eLife.29257