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Frontiers in Synaptic Neuroscience 2023Cannabis exposure during gestation evokes significant molecular modifications to neurodevelopmental programs leading to neurophysiological and behavioral abnormalities...
Cannabis exposure during gestation evokes significant molecular modifications to neurodevelopmental programs leading to neurophysiological and behavioral abnormalities in humans. The main neuronal receptor for Δ-tetrahydrocannabinol (THC) is the type-1 cannabinoid receptor CBR, one of the most abundant G-protein-coupled receptors in the nervous system. While THC is the major psychoactive phytocannabinoid, endocannabinoids (eCBs) are the endogenous ligands of CBR and are known to act as retrograde messengers to modulate synaptic plasticity at different time scales in the adult brain. Accumulating evidence indicates that eCB signaling through activation of CBR plays a central role in neural development. During development, most CBR localized to axons of projection neurons, and in mice eCB signaling impacts axon fasciculation. Understanding of eCB-mediated structural plasticity during development, however, requires the identification of the precise spatial and temporal dynamics of CBR-mediated modifications at the level of individual neurons in the intact brain. Here, the cell-autonomous role of CBR and the effects of CBR-mediated eCB signaling were investigated using targeted single-cell knockdown and pharmacologic treatments in . We imaged axonal arbors of retinal ganglion cells (RGCs) in real time following downregulation of CBR morpholino (MO) knockdown. We also analyzed RGC axons with altered eCB signaling following treatment with URB597, a selective inhibitor of the enzyme that degrades Anandamide (AEA), or JZL184, an inhibitor of the enzyme that blocks 2-Arachidonoylglycerol (2-AG) hydrolysis, at two distinct stages of retinotectal development. Our results demonstrate that CBR knockdown impacts RGC axon branching at their target and that differential 2-AG and AEA-mediated eCB signaling contributes to presynaptic structural connectivity at the time that axons terminate and when retinotectal synaptic connections are made. Altering CBR levels through CBR MO knockdown similarly impacted dendritic morphology of tectal neurons, thus supporting both pre- and postsynaptic cell-autonomous roles for CBR-mediated eCB signaling.
PubMed: 37252636
DOI: 10.3389/fnsyn.2023.1176864 -
ELife Oct 2022Development of elaborate and polarized neuronal morphology requires precisely regulated transport of cellular cargos by motor proteins such as kinesin-1. Kinesin-1 has...
Development of elaborate and polarized neuronal morphology requires precisely regulated transport of cellular cargos by motor proteins such as kinesin-1. Kinesin-1 has numerous cellular cargos which must be delivered to unique neuronal compartments. The process by which this motor selectively transports and delivers cargo to regulate neuronal morphogenesis is poorly understood, although the cargo-binding kinesin light chain (KLC) subunits contribute to specificity. Our work implicates one such subunit, KLC4, as an essential regulator of axon branching and arborization pattern of sensory neurons during development. Using live imaging approaches in mutant zebrafish, we show that KLC4 is required for stabilization of nascent axon branches, proper microtubule (MT) dynamics, and endosomal transport. Furthermore, KLC4 is required for proper tiling of peripheral axon arbors: in mutants, peripheral axons showed abnormal fasciculation, a behavior characteristic of central axons. This result suggests that KLC4 patterns axonal compartments and helps establish molecular differences between central and peripheral axons. Finally, we find that mutant larva are hypersensitive to touch and adults show anxiety-like behavior in a novel tank test, implicating as a new gene involved in stress response circuits.
Topics: Animals; Kinesins; Zebrafish; Axons; Sensory Receptor Cells; Morphogenesis
PubMed: 36222498
DOI: 10.7554/eLife.74270 -
Neuroscience Insights 2022During nervous system development, axons must navigate to specific target areas. In , the cadherin CDH-4 is required for ventral nerve cord axonal navigation, and dorsal...
During nervous system development, axons must navigate to specific target areas. In , the cadherin CDH-4 is required for ventral nerve cord axonal navigation, and dorsal nerve cord fasciculation. How CDH-4 mediates axon navigation and fasciculation is currently unknown. To identify genes acting together with , we isolated mutants suppressing the axon guidance defects of mutants. These suppressors showed partial suppression of axonal defects in the dorsal and ventral nerve cords seen in mutants. We identified one suppressor gene, , which encodes a component of the spliceosome. Complete loss-of-function alleles of are lethal, suggesting that the mutation isolated in our suppressor screen is a partial loss-of-function allele. A previous study found that RNAi-induced suppression of leads to changes in the expression of several 100 genes including the cadherin . We found that overexpression of mimics the suppression seen in mutants, suggesting that CDH-5 can partially compensate for the loss of CDH-4.
PubMed: 36090596
DOI: 10.1177/26331055221123346 -
Cells Aug 2022Axonal varicosities or swellings are enlarged structures along axon shafts and profoundly affect action potential propagation and synaptic transmission. These...
Axonal varicosities or swellings are enlarged structures along axon shafts and profoundly affect action potential propagation and synaptic transmission. These structures, which are defined by morphology, are highly heterogeneous and often investigated concerning their roles in neuropathology, but why they are present in the normal brain remains unknown. Combining confocal microscopy and cryo-electron tomography (Cryo-ET) with in vivo and in vitro systems, we report that non-uniform mechanical interactions with the microenvironment can lead to 10-fold diameter differences within an axon of the central nervous system (CNS). In the brains of adult Thy1-YFP transgenic mice, individual axons in the cortex displayed significantly higher diameter variation than those in the corpus callosum. When being cultured on lacey carbon film-coated electron microscopy (EM) grids, CNS axons formed varicosities exclusively in holes and without microtubule (MT) breakage, and they contained mitochondria, multivesicular bodies (MVBs), and/or vesicles, similar to the axonal varicosities induced by mild fluid puffing. Moreover, enlarged axon branch points often contain MT free ends leading to the minor branch. When the axons were fasciculated by mimicking in vivo axonal bundles, their varicosity levels reduced. Taken together, our results have revealed the extrinsic regulation of the three-dimensional ultrastructures of central axons by the mechanical microenvironment under physiological conditions.
Topics: Action Potentials; Animals; Axons; Corpus Callosum; Electron Microscope Tomography; Mice; Microtubules
PubMed: 36010609
DOI: 10.3390/cells11162533 -
Frontiers in Integrative Neuroscience 2022Mounting evidence supports a key involvement of the chondroitin sulfate proteoglycans (CSPGs) NG2 and brevican (BCAN) in the regulation of axonal functions, including...
Mounting evidence supports a key involvement of the chondroitin sulfate proteoglycans (CSPGs) NG2 and brevican (BCAN) in the regulation of axonal functions, including axon guidance, fasciculation, conductance, and myelination. Prior work suggested the possibility that these functions may, at least in part, be carried out by specialized CSPG structures surrounding axons, termed axonal coats. However, their existence remains controversial. We tested the hypothesis that NG2 and BCAN, known to be associated with oligodendrocyte precursor cells, form axonal coats enveloping myelinated axons in the human brain. In tissue blocks containing the mediodorsal thalamic nucleus (MD) from healthy donors ( = 5), we used dual immunofluorescence, confocal microscopy, and unbiased stereology to characterize BCAN and NG2 immunoreactive (IR) axonal coats and measure the percentage of myelinated axons associated with them. In a subset of donors ( = 3), we used electron microscopy to analyze the spatial relationship between axons and NG2- and BCAN-IR axonal coats within the human MD. Our results show that a substantial percentage (∼64%) of large and medium myelinated axons in the human MD are surrounded by NG2- and BCAN-IR axonal coats. Electron microscopy studies show NG2- and BCAN-IR axonal coats are interleaved with myelin sheets, with larger axons displaying greater association with axonal coats. These findings represent the first characterization of NG2 and BCAN axonal coats in the human brain. The large percentage of axons surrounded by CSPG coats, and the role of CSPGs in axonal guidance, fasciculation, conductance, and myelination suggest that these structures may contribute to several key axonal properties.
PubMed: 35875507
DOI: 10.3389/fnint.2022.934764 -
Seminars in Cell & Developmental Biology May 2023Neural networks are constructed through the development of robust axonal projections from individual neurons, which ultimately establish connections with their targets.... (Review)
Review
Neural networks are constructed through the development of robust axonal projections from individual neurons, which ultimately establish connections with their targets. In most animals, developing axons assemble in bundles to navigate collectively across various areas within the central nervous system or the periphery, before they separate from these bundles in order to find their specific targets. These processes, called fasciculation and defasciculation respectively, were thought for many years to be controlled chemically: while guidance cues may attract or repulse axonal growth cones, adhesion molecules expressed at the surface of axons mediate their fasciculation. Recently, an additional non-chemical parameter, the mechanical longitudinal tension of axons, turned out to play a role in axon fasciculation and defasciculation, through zippering and unzippering of axon shafts. In this review, we present an integrated view of the currently known chemical and mechanical control of axon:axon dynamic interactions. We highlight the facts that the decision to cross or not to cross another axon depends on a combination of chemical, mechanical and geometrical parameters, and that the decision to fasciculate/defasciculate through zippering/unzippering relies on the balance between axon:axon adhesion and their mechanical tension. Finally, we speculate about possible functional implications of zippering-dependent axon shaft fasciculation, in the collective migration of axons, and in the sorting of subpopulations of axons.
Topics: Animals; Fasciculation; Axon Fasciculation; Axons; Neurons; Central Nervous System
PubMed: 35810068
DOI: 10.1016/j.semcdb.2022.06.014 -
Frontiers in Neuroscience 2022Precise wiring of neural circuits is essential for brain connectivity and function. During development, axons respond to diverse cues present in the extracellular matrix... (Review)
Review
Precise wiring of neural circuits is essential for brain connectivity and function. During development, axons respond to diverse cues present in the extracellular matrix or at the surface of other cells to navigate to specific targets, where they establish precise connections with post-synaptic partners. Cell adhesion molecules (CAMs) represent a large group of structurally diverse proteins well known to mediate adhesion for neural circuit assembly. Through their adhesive properties, CAMs act as major regulators of axon navigation, fasciculation, and synapse formation. While the adhesive functions of CAMs have been known for decades, more recent studies have unraveled essential, non-adhesive functions as well. CAMs notably act as guidance cues and modulate guidance signaling pathways for axon pathfinding, initiate contact-mediated repulsion for spatial organization of axonal arbors, and refine neuronal projections during circuit maturation. In this review, we summarize the classical adhesive functions of CAMs in axonal development and further discuss the increasing number of other non-adhesive functions CAMs play in neural circuit assembly.
PubMed: 35573298
DOI: 10.3389/fnins.2022.889155 -
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 -
The European Journal of Neuroscience Feb 2022Previous studies show that the main cannabinoid receptor in the brain-cannabinoid type 1 receptor (CB1R)-is required for establishment of axonal projections in...
Previous studies show that the main cannabinoid receptor in the brain-cannabinoid type 1 receptor (CB1R)-is required for establishment of axonal projections in developing neurons but questions remain regarding the cellular and molecular mechanisms, especially in neurons developing in their native environment. We assessed the effects of CB1R signalling on growth cone filopodia and axonal projections of retinal ganglion cells (RGCs) in whole mount brains from Xenopus laevis tadpoles. Our results indicate that growth cones of RGC axons in brains from tadpoles exposed to a CB1R agonist had fewer filopodial protrusions, whereas growth cones from tadpoles exposed to a CB1R inverse agonist had more filopodia than growth cones of RGC axons in whole brains from control tadpoles. However, application of both the CB1R agonist and inverse agonist resulted in RGC axons that were overly dispersed and undulatory in the optic tract in situ. In addition, expression of a mutant for cadherin adhesive factor, β-catenin, that disrupts its binding to α-catenin, and application of an inhibitor for actin regulator non-muscle Myosin II, phenocopied the effects of the CB1R agonist and inverse agonist on growth cone filopodia, respectively. These findings suggest that both destablization and stabilization of growth cone filopodia are required for RGC axonal fasciculation/defasciculation in the optic tract and that CB1R regulates growth cone filopodia and axon dispersion of RGCs by oppositely modulating β-catenin adhesive and Myosin II actin regulatory functions. This study extends and confirms our understanding of cannabinoid mechanisms in sculpting developing neuronal circuits in vivo.
Topics: Actins; Animals; Axons; Cannabinoids; Growth Cones; Larva; Optic Tract; Pseudopodia; Receptors, Cannabinoid; Retinal Ganglion Cells; Xenopus laevis; beta Catenin
PubMed: 35060216
DOI: 10.1111/ejn.15603 -
Brain : a Journal of Neurology Apr 2022Understanding new modulators of axon regeneration is central to neural repair. Our previous work demonstrated critical roles of atypical cadherin Celsr2 during neural...
Understanding new modulators of axon regeneration is central to neural repair. Our previous work demonstrated critical roles of atypical cadherin Celsr2 during neural development, including cilia organization, neuron migration and axon navigation. Here, we address its role in axon regeneration. We show that Celsr2 is highly expressed in both mouse and human spinal motor neurons. Celsr2 knockout promotes axon regeneration and fasciculation in mouse cultured spinal explants. Similarly, cultured Celsr2 mutant motor neurons extend longer neurites and larger growth cones, with increased expression of end-binding protein 3 and higher potassium-induced calcium influx. Mice with Celsr2 conditional knockout in spinal motor neurons do not exhibit any behavioural deficits; however, after branchial plexus injury, axon regeneration and functional forelimb locomotor recovery are significantly improved. Similarly, knockdown of CELSR2 using shRNA interference in cultured human spinal motor explants and motor neurons increases axonal fasciculation and growth. In mouse adult spinal cord after root avulsion, in mouse embryonic spinal cords, and in cultured human motor neurons, Celsr2 downregulation is accompanied by increased levels of GTP-bound Rac1 and Cdc42, and of JNK and c-Jun. In conclusion, Celsr2 negatively regulates motor axon regeneration and is a potential target to improve neural repair.
Topics: Animals; Axon Fasciculation; Axons; Cadherins; Humans; Mice; Motor Neurons; Nerve Regeneration; Spinal Cord; Spinal Cord Injuries
PubMed: 34983065
DOI: 10.1093/brain/awab317