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Growth Cones

Q: What are the key functions of growth cones in neuronal development?

Growth cones are crucial structures at the tips of developing axons and dendrites. They play a vital role in guiding the extension and path-finding of the growing neurite. Growth cones sense environmental cues and direct the movement of the axon or dendrite, which is essential for the proper wiring and connectivity of the nervous system during development.

Q: How do growth cones facilitate neuronal motility and response to guidance signals?

Growth cones contain actin filaments and microtubules that enable their dynamic movements and ability to respond to various guidance signals. These cytoskeletal components facilitate the motility and adaptability of growth cones, allowing them to navigate through the developing environment and steer the growing neurite towards its target.

Growth cones are specialized, dynamic structures found at the tips of developing axons and dendrites.
These structures play a crucial role in the guidance and navigation of the growing neurite, sensing environmental cues and directing the extension and path-finding of the axon or dendrite.
Growth cones are essential for the proper wiring and connectivity of the nervous system during development.
They contain actin filaments and microtubules that facilitate their motility and ability to respond to guidance signals.
Understanding the mechanisms regulating growth cone dynamics and behavior is a key area of neuroscience reseach, as it provides insights into neuronal development and potential targets for therapies addressing neurodevelopmental disorders.
PubCompare.ai can help researchers easily identify the best protocols and products to optimize their growth cone research.

Most cited protocols related to «Growth Cones»

Mical Regulates Actin and Microtubule Dynamics

We performed complementation analysis, genetics, molecular biology, western blotting, immunostaining and generation of transgenic animals using standard techniques4 (link). Multiple new lines of the full-length ‘short’ isoform of Mical, the Mical^Δredox mutation (Mical^G→W; ref. 4 (link)) and the other transgenic animals were generated and used for all experiments. Adult bristles were examined and quantified by crossing adults at 25 °C: adult offspring from these crosses were first sorted according to genotype and then examined under a dissecting microscope. We genotyped pupae using a Zeiss Discovery M² Bio fluorescence stereomicroscope, and all preparation, staging and dissection of pupae were done using standard approaches. We imaged, drew and quantified the adult bristles with the aid of the Discovery M² Bio stereomicroscope, a motorized focus and zoom, a Zeiss AxioCam HR camera and three-dimensional-reconstruction software (Zeiss AxioVision, version 4.6.3, and Extended Focus software). All other bright-field, dark-field, differential interference contrast and fluorescence visualization, and imaging of bristles, embryos and growth cones, was done using a Zeiss Axio Imager upright microscope with motorized focus and zoom and an ApoTome module, and images were captured and quantified using the AxioCam HR camera and AxioVision software. All electron microscopy of pupae and negative staining of purified proteins was done using a FEI Tecnai G² Spirit BioTWIN transmission electron microscope. We purified recombinant Mical proteins10 and recombinant p-hydroxybenzoate hydroxylase using our previously developed approaches. Drosophila fascin (also known as singed) complementary DNA was inserted in a bacterial expression vector, and recombinant Drosophila fascin protein was purified. All F-actin and Mical co-sedimentation assays and G-actin/F-actin ratio experiments were performed using standard approaches, as were all pyrene-labelled actin polymerization and depolymerization assays, actin bundling assays, tubulin polymerization assays and microtubule co-sedimentation assays.

Hung R.J., Yazdani U., Yoon J., Wu H., Yang T., Gupta N., Huang Z., van Berkel W.J, & Terman J.R. (2010). Mical links semaphorins to F-actin disassembly. Nature, 463(7282), 823-827.

Publication 2010

Actins Adult Animals, Transgenic Bacteria Biological Assay Cloning Vectors Dissection DNA, Complementary Drosophila Electron Microscopy Embryo F-Actin fascin Fluorescence G-Actin Growth Cones Hydroxybenzoates Microscopy Microtubules Mixed Function Oxygenases Mutation Polymerization Protein Isoforms Proteins Pupa Pyrenes Reconstructive Surgical Procedures Sn protein, Drosophila Transmission Electron Microscopy Tubulin

PFN1 Mutant Characterization and Function

Exome sequencing was accomplished using an exome array (SeqCap EZ Exome Library, Nimblegen) adapted for sequencing on the Illumina DNA sequencing platform. Alignment of sequence to the human genome and variant detection was accomplished using the applications SOAPaligner and SOAPsnp. Expression constructs for wild-type and mutant PFN1 were transfected into N2A using Lipofectamine 2000 (Invitrogen). Inhibition of proteasome activity in N2A cells was performed by incubation with 10 μM MG132 (Sigma-Aldrich) after transfection for 16 hours before collection. Insolubility of PFN1 mutants was assessed by sequential NP-40/urea protein extraction followed by western blot analysis. Transfected HEK293 cells were lysed at 24 hours with RIPA buffer and immunoprecitated with an anti-V5 antibody to investigate the interactions of PFN1 and actin. Primary motor neurons were isolated and cultured from E13.5 mouse embryos and transfected by magnetofection. Axon length measurements were determined from low magnification images (10x). The length of the longest axon branch was measured using ImageJ plug-in NeuronJ. F-actin and G-actin were labeled with fluorescent-conjugated Phalloidin (Invitrogen) and DNase I (Invitrogen), respectively. Deconvolution of images was performed using Autoquant (MediaCybernetics). The growth cone area and the fluorescence intensity for F-actin and G-actin staining was measured using ImageJ software.

Wu C.H., Fallini C., Ticozzi N., Keagle P.J., Sapp P.C., Piotrowska K., Lowe P., Koppers M., McKenna-Yasek D., Baron D.M., Kost J.E., Gonzalez-Perez P., Fox A.D., Adams J., Taroni F., Tiloca C., Leclerc A.L., Chafe S.C., Mangroo D., Moore M.J., Zitzewitz J.A., Xu Z.S., van den Berg L.H., Glass J.D., Siciliano G., Cirulli E.T., Goldstein D.B., Salachas F., Meininger V., Rossoll W., Ratti A., Gellera C., Bosco D.A., Bassell G.J., Silani V., Drory V.E., Brown RH J.r, & Landers J.E. (2012). Mutations in the Profilin 1 Gene Cause Familial Amyotrophic Lateral Sclerosis. Nature, 488(7412), 499-503.

Publication 2012

Actins Antibodies, Anti-Idiotypic Axon Buffers Cells Deoxyribonuclease I DNA Library Embryo Exome F-Actin Fluorescence G-Actin Genome, Human Growth Cones HEK293 Cells lipofectamine 2000 MG 132 Motor Neurons Multicatalytic Endopeptidase Complex Mus Nonidet P-40 Phalloidine Proteins Psychological Inhibition Radioimmunoprecipitation Assay Sequence Alignment Transfection Urea Western Blot

PFN1 Mutant Characterization and Function

Publication 2012

Quantifying Mitochondrial Membrane Potential

For determination of mitochondrial transmembrane potential (Scheffler, 1999 ; Nicholls and Budd, 2000 (link)), a 5 μM stock of tetramethylrhodamine methyl ester (TMRM, Molecular Probes) in DMSO was diluted into F+ medium to a final concentration of 20 nM. Cultures were exposed to 20 nM TMRM for 20 min at 37°C to allow dye equilibration across the plasma and inner mitochondrial membranes (Johnson et al., 1980 (link); Farkas et al., 1989 (link); Scaduto and Grotyohann, 1999 (link)). To confirm the rate of equilibration of TMRM across the plasma membrane, cytoplasmic fluorescence was measured in axons and growth cones after incubation in 20 nM dye for times ranging from 2 to 20 min. In both regions cytoplasmic fluorescence reached its peak level within 2 min and increased no further, indicating that a 20 min incubation is more than adequate for dye equilibration. The matrix TMRM concentration of cells exposed in this way is subquenching, resulting in a direct relationship between fluorescence intensity and ΔΨ_m (Ward et al., 2000 (link); Nakayama et al., 2002 (link)). For imaging, the medium was replaced with F+ containing 5 nM TMRM. Mitotracker Green FM (Molecular Probes) was stored as a stock in DMSO and diluted to a final concentration of 50 nM in F+ medium along with 20 nM TMRM. Cells were incubated in this mixture for 15 min before being transferred to F+ medium with 20 nM TMRM but no Mitotracker and incubated for 5 min more to rinse out excess MitoTracker. For microscopy, cells were transferred to F+ with 5 nM TMRM. JC-1 staining was carried out as previously described (Overly et al., 1996 (link)).

Verburg J, & Hollenbeck P.J. (2008). Mitochondrial Membrane Potential in Axons Increases with Local Nerve Growth Factor or Semaphorin Signaling. The Journal of Neuroscience, 28(33), 8306-8315.

Publication 2008

Axon Cells Cytoplasm Cytosol Fluorescence Growth Cones Membrane Potential, Mitochondrial Microscopy Mitochondrial Membrane, Inner mitotracker green FM Molecular Probes Plasma Plasma Membrane Sulfoxide, Dimethyl tetramethylrhodamine methyl ester

Selective Axon Manipulation in DRG Neurons

To apply NGF, translation inhibitors, or siRNA specifically to distal axons and growth cones without affecting the cell bodies, E14 dissociated DRG neurons were grown in microfluidic chambers with 450-µm-long microgrooves14 (link) (Xona Microfluidics, Temecula, CA). These chambers allow fluidic isolation of the axonal compartment from the cell body compartment14 (link). The microfluidic chambers were coated with 100 µg ml⁻¹ poly-L-lysine (Trevigen, Gaithersburg, MD). The growth medium was completely exchanged after 48 h. siRNA transfection in the axonal compartment was performed on DIV3, and outgrowth assays and FISH were done on DIV4.

Hengst U., Deglincerti A., Kim H.J., Jeon N.L, & Jaffrey S.R. (2009). Axonal elongation triggered by stimulus-induced local translation of a polarity complex protein. Nature cell biology, 11(8), 1024-1030.

Publication 2009

Axon Biological Assay Body Fluid Compartments Cell Body Culture Media Fishes Growth Cones isolation Lysine Neurons Poly A Protein Synthesis Inhibitors RNA, Small Interfering Transfection

Most recents protocols related to «Growth Cones»

Statistical Analysis of Neuromuscular Junction and Growth Cone

Statistical analysis was performed using the software programs MS Excel 2016 (Microsoft, Redmond, WA, USA) and GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). Unpaired, two tailed Student’s t-tests and one-way ANOVA statistics tests with Tukey post hoc test for multiple comparisons were performed. To compare categories (NMJ innervation and growth cone analysis) χ² test was performed, and for more than two comparisons, for normalization, the p value was multiplied by the total number of comparisons. The figure legends depict the specific statistical test used, sample size and p-values, respectively.

Muiños-Bühl A., Rombo R., Ling K.K., Zilio E., Rigo F., Bennett C.F, & Wirth B. (2023). Long-Term SMN- and Ncald-ASO Combinatorial Therapy in SMA Mice and NCALD-ASO Treatment in hiPSC-Derived Motor Neurons Show Protective Effects. International Journal of Molecular Sciences, 24(4), 4198.

Publication 2023

Growth Cones neuro-oncological ventral antigen 2, human prisma Student

Laser Stimulation of Cell Dynamics

The cells were stimulated with a laser spot whose full width at half maximum was 3

μ

m on the image plane. The superficial power density was 5.7 mW

/ {μ m}^{2},

if absorption is neglected, which is a justified assumption [22 (link)]. The laser was intentionally slightly out of focus to prevent optical trapping phenomena. Its position was controlled using the additional set of Galvo mirrors at the rear input of the microscope. The center of the laser spot was located 5 µm away from the leading edge of the growth cone or the lamellipodium. The position of the laser was adjusted to a few angular degrees above or below the forward direction when the leading edge came close to the laser. The optical stimulation experiments consisted of a 20 min period without laser stimulation (PREV), followed by two cycles of 20 min with the laser (ON) and 20 min without the laser (OFF). This sequence was adopted for both cell lines, PC12 and 3T3. For fluorescence images of the cytoskeleton, samples were recorded for several sequences.

Medina-Villalobos N., Avila R., Marsal M., Andilla J., Loza-Álvarez P., Ojeda-Ramírez M.M, & Tamariz E. (2023). Infrared Laser Effects on Cell Projection Depend on Irradiation Intermittence and Cell Activity. Cells, 12(4), 540.

Publication 2023

Cell Lines Cells Cytoskeleton Fluorescence Growth Cones Lamellipodia Microscopy Optical Phenomena PC12 Cells Photic Stimulation

Detailed Neuronal Morphology Analysis

For neurite tracing and Sholl analysis, neurites and axons were traced and then measured using the Simple Neurite Tracer (SNT) plugin in Fiji ImageJ. Axons were defined as β-III tubulin⁺Map2⁻ neurites. Thy1-YFP⁺ pyramidal neurons in the layer V of motor cortex were traced with SNT in ImageJ. In vivo 3D neuronal Sholl analysis was performed on basal dendrites, the radius increment was set at 10 μm. Axon growth cone size was determined in Fiji ImageJ by manually tracing and measuring the area of regions of interest (ROIs) based on the anti-GAP43 antibody fluorescence at the tip of β-III tubulin⁺ (or Tau⁺) Map2⁻ axons. For quantification of VGluT1 or PSD95 puncta, confocal images were taken using the Leica SPE confocal laser scanning microscope (9–12 μm Z-stack with 0.5 μm step) magnified with 63x objective and first converted to projection images (with maximal projection) for analyses. The software SynPAnal 2 was used for quantifying the puncta density and intensity/area of PSD95⁺ and VGLUT1⁺ puncta. Neurite segments (20–30 μm in length) were quantified from each neuron and their average values were also measured using SynPAnal software.
For extracellular and intracellular CD63-GFP⁺ puncta analysis, the extracellular percentage ratio of CD63-GFP⁺ puncta were determined in relation to the tdT⁺ astroglia using Fiji ImageJ based on confocal images. The CD63-GFP channel image was first thresholded to create a binary black and red image. Then the Measure Analyzer tool was used to count all CD63-GFP⁺ puncta area. The tdT channel image was thresholded and the Particle Analyzer tool was used to generate the ROIs of all tdT⁺ signals. Then the ROIs of tdT⁺ signals were overlaid on the CD63-GFP⁺ images. CD63-GFP⁺ area was then measured inside of tdT based ROIs. CD63-GFP⁺ puncta inside tdT⁺ ROIs were considered as intracellular CD63-GFP⁺ signals. Extracellular CD63-GFP⁺ area was determined by subtracting CD63-GFP⁺ intracellular area from total CD63-GFP⁺ area and the extracellular percentage ratio was calculated by dividing the total CD63-GFP⁺ area by the extracellular CD63⁺ area.
For quantification of dendritic spine density, confocal images of eGFP⁺ pyramidal neurons of layer V motor cortex of Thy1-YFP⁺ and Thy1-YFP⁺ApoE^−/− mice were acquired at 0.5 μm intervals with a 63×oil immersion lens with Leica falcon confocal microscope. 3D reconstruction of eGFP⁺ neurons was built using the Imaris image analysis software (Bitplane). Both apical collateral and basal dendrites and spines were traced with the filament tracing function in Imaris and quantified. The dendritic spine density was calculated by dividing the number of spines by dendrite length (~30 to 40 μm).

Jin S., Chen X., Tian Y., Jarvis R., Promes V, & Yang Y. (2023). Astroglial exosome HepaCAM signaling and ApoE antagonization coordinates early postnatal cortical pyramidal neuronal axon growth and dendritic spine formation. bioRxiv.

Publication Preprint 2023

Apolipoproteins E Astrocytes Axon Cytoskeletal Filaments Dendrites Dendritic Spines Fluorescent Antibody Technique Growth Cones Lens, Crystalline MAP2 protein, human Mice, Laboratory Microscopy, Confocal Microscopy, Confocal, Laser Scanning Motor Cortex Neurites Neurons Protoplasm Pyramidal Cells Radius Reconstructive Surgical Procedures Submersion Tubulin Vertebral Column

Culturing Dopaminergic Neurons and ReNcell VM Cells

The SH-SY5Y cell line was a kind gift from Shi Yun (Animal Research Center, Nanjing University, China). Cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum at 37 °C in 5% CO₂.
Primary dopaminergic neuron cultures were prepared from the ventral mesencephalon of embryonic day 13.5 (E13.5) fetal mice. Briefly, embryos were obtained and placed in Hanks’ balanced salt solution and dissected to obtain mesencephalon tissue. Next, it was treated with 0.125% trypsin for 5 min at 37 °C, followed by neurobasal medium supplemented with 10% FBS. Approximately 2 × 10⁵ cells/mL were seeded on poly-L-lysine-coated coverslips and cultured in 500 μL of neurobasal medium supplemented with 2% B27 and 0.5 mM glutamine. The medium was replaced with fresh medium after 24 h and the cells were cultured for 7 days before incubation with 200 μM MPP⁺ for 24 h. Then, the cells were characterized by immunostaining for Tuj1 and TH.
ReNcell VM cells (purchased from Millipore) were cultured as neurospheres in DMEM/F12 supplemented with 2% B27, 20 ng/mL epidermal growth factor (EGF), and 20 ng/mL basic fibroblast growth factor (bFGF). To label ReNcell with tdTomato, cells were stably transduced with packaged lentivirus vectors to express tdTomato fused with the palmitoylation sequence of growth cone-associated protein (PalmtdTomato). The plasmid was kindly provided by Dr. Bakhos Tannous (Massachusetts General Hospital, Boston, MA, USA). After 5–7 days of proliferation, aggregated cells were collected and dissociated by gentle trituration and replated on laminin-coated coverslips with media without bFGF and EGF. Differentiation was initiated by the addition of 1 mM dibutyryl-cAMP and 2 ng/mL glial cell-derived neurotrophic factor (GDNF). Experiments were conducted 12 days after differentiation.

Wang J., Xu S.Y., Ye Z.Y., Sun Z.N., Zhang J.Q., Qi C., Liu R., Gao X., He C., You W.Y, & Gao J. (2023). The deficiency of Maged1 attenuates Parkinson's disease progression in mice. Molecular Brain, 16, 22.

Publication 2023

Amino Acid Sequence Cell Lines Cells Cloning Vectors Dopaminergic Neurons Embryo Epidermal growth factor Fetus Fibroblast Growth Factor 2 Glial Cell Line-Derived Neurotrophic Factor Glutamine Growth Cones Hanks Balanced Salt Solution Laminin Lentivirus Lysine Mesencephalon Mus Palmitoylation Plasmids Poly A tdTomato Tissues Trypsin

Live Imaging of i³Neuron Dynamics

i³Neuron imaging experiments were performed 1–3 days after replating on laminin-coated glass-bottom dishes. At later times i³Neurons form intricate networks and it becomes increasingly difficult to locate individual growth cones. SPY555-tubulin and SPY650-FastAct (Spyrochrome) were added to i³Neurons at a 1:2000 and 1:3000 dilution, respectively, and 4–610 CP-CTX was added at 5 nM for at least 30 min before imaging, and cells were discarded after a maximum of 3 hr.
Live microscopy was performed either with a Yokogawa CSU-X1 spinning disk confocal essentially as described (Stehbens et al., 2012 (link)) or, for most i³Neuron microscopy, with a CFI Apochromat TIRF 60 X NA 1.49 objective (Nikon) on a Yokogawa CSU-W1/SoRa spinning disk confocal system, and images acquired with an ORCA Fusion BT sCMOS camera (Hamamatsu). For high-resolution imaging of dim signal, SoRa mode was combined with 2x2 camera binning resulting in an image pixel size of 54 nm. This system was equipped with a Polygon 1000 pattern illuminator (Mightex) through an auxiliary filter turret and LAPP illuminator (Nikon). Integrated control of imaging and photoinactivation was through NIS Elements v5.3 software (Nikon), in combination with an external pulse generator to trigger the 470 nm photoinactivation LED (Spectra X light engine, Lumencor) for 10 ms pulses at 2 Hz at 5–10% LED power. π-EB1 photoinactivation was confirmed by dissociation of the C-terminal half from growing MT ends. The Polygon was calibrated with a mirror slide before every imaging session, and local light exposure was further verified by imaging the back reflection from the specimen coverslip.

Dema A., Charafeddine R., Rahgozar S., van Haren J, & Wittmann T. (2023). Growth cone advance requires EB1 as revealed by genomic replacement with a light-sensitive variant. eLife, 12, e84143.

Publication 2023

Cells Growth Cones Hyperostosis, Diffuse Idiopathic Skeletal Laminin Light Microscopy Orcinus orca Precipitating Factors Pulse Rate Reflex Technique, Dilution Tubulin

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Neurobasal medium is a cell culture medium designed for the maintenance and growth of primary neuronal cells. It provides a defined, serum-free environment that supports the survival and differentiation of neurons. The medium is optimized to maintain the phenotypic characteristics of neurons and minimizes the growth of non-neuronal cells.

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What are the key functions of growth cones in neuronal development?

How do growth cones facilitate neuronal motility and response to guidance signals?

What are some common challenges in optimizing growth cone research protocols?

Researchers often face challenges in identifying the most effective protocols and products for their growth cone research. Sifting through the vast amount of literature, pre-prints, and patents to find the optimal methods can be time-consuming and tedious. Additionally, understanding the nuances and key differences between various protocols can be difficult, impacting the reproducibility and accuracy of the research.

How can PubCompare.ai assist in growth cone research?

PubCompare.ai can help researchers optimize their growth cone research protocols. The platform's AI-driven analysis allows you to more efficiently screen protocol literature and pinpoint critical insights. PubCompare.ai can highlight key differences in protocol effectiveness, enabling you to choose the best option for your specific research goals and ensure reproducibility and accuaracy. This can save you time and effort in identifying the most effective methods and products for your growth cone studies.

More about "Growth Cones"

Growth cones are specialized, dynamic structures found at the tips of developing axons and dendrites.
These structures, also known as neuronal growth cones or neurite growth cones, play a crucial role in the guidance and navigation of the growing neurite.
They are essential for the proper wiring and connectivity of the nervous system during development.
Growth cones contain actin filaments and microtubules that facilitate their motility and ability to respond to guidance signals, such as chemical cues in the extracellular environment.
These dynamic structures sense and interpret environmental signals, directing the extension and path-finding of the axon or dendrite.
Understanding the mechanisms regulating growth cone dynamics and behavior is a key area of neuroscience research, as it provides insights into neuronal development and potential targets for therapies addressing neurodevelopmental disorders.
Researchers often utilize tools like Volocity, Rhodamine-conjugated phalloidin, Axio Observer, Neurobasal medium, LSM 510 META, Texas Red, Rolera mGi camera, LSM 710, and LSM Image Browser to visualize and analyze growth cone structure and function.
By optimizing growth cone research protocols, scientists can gain a deeper understanding of how these specialized structures guide the development of the nervous system and identify new avenues for interventions targeting neuronal connectivity and development.
PubCompare.ai can help researchers easily identify the best protocols and products to advance their growth cone research.