RESUMEN
Geometrical features play a very important role in neuronal growth and the formation of functional connections between neuronal cells. Here, we analyze the dynamics of axonal growth for neuronal cells cultured on micro-patterned polydimethylsiloxane surfaces. We utilize fluorescence microscopy to image axons, quantify their dynamics, and demonstrate that periodic geometrical patterns impart strong directional bias to neuronal growth. We quantify axonal alignment and present a general stochastic approach that quantitatively describes the dynamics of the growth cones. Neuronal growth is described by a general phenomenological model, based on a simple automatic controller with a closed-loop feedback system. We demonstrate that axonal alignment on these substrates is determined by the surface geometry, and it is quantified by the deterministic part of the stochastic (Langevin and Fokker-Planck) equations. We also show that the axonal alignment with the surface patterns is greatly suppressed by the neuron treatment with Blebbistatin, a chemical compound that inhibits the activity of myosin II. These results give new insight into the role played by the molecular motors and external geometrical cues in guiding axonal growth, and could lead to novel approaches for bioengineering neuronal regeneration platforms.
Asunto(s)
Dimetilpolisiloxanos , Neurogénesis , Neuronas/fisiología , Polilisina , Animales , Células Cultivadas , Microscopía de Fuerza Atómica , Microscopía Fluorescente , RatasRESUMEN
Detailed knowledge of how the surface physical properties, such as mechanics, topography and texture influence axonal outgrowth and guidance is essential for understanding the processes that control neuron development, the formation of functional neuronal connections and nerve regeneration. Here we synthesize asymmetric surfaces with well-controlled topography and texture and perform a systematic experimental and theoretical investigation of axonal outgrowth on these substrates. We demonstrate unidirectional axonal bias imparted by the surface ratchet-based topography and quantify the topographical guidance cues that control neuronal growth. We describe the growth cone dynamics using a general stochastic model (Fokker-Planck formalism) and use this model to extract two key dynamical parameters: diffusion (cell motility) coefficient and asymmetric drift coefficient. The drift coefficient is identified with the torque caused by the asymmetric ratchet topography. We relate the observed directional bias in axonal outgrowth to cellular contact guidance behavior, which results in an increase in the cell-surface coupling with increased surface anisotropy. We also demonstrate that the disruption of cytoskeletal dynamics through application of Taxol (stabilizer of microtubules) and Blebbistatin (inhibitor of myosin II activity) greatly reduces the directional bias imparted by these asymmetric surfaces. These results provide new insight into the role played by topographical cues in neuronal growth and could lead to new methods for stimulating neuronal regeneration and the engineering of artificial neuronal tissue.
Asunto(s)
Axones/fisiología , Citoesqueleto/metabolismo , Modelos Biológicos , Paclitaxel/farmacología , Regeneración/efectos de los fármacos , Animales , Células Cultivadas , Ratas , Procesos EstocásticosRESUMEN
Current understanding of neuronal growth is mostly qualitative, as the staggering number of physical and chemical guidance cues involved prohibit a fully quantitative description of axonal dynamics. We report on a general approach that describes axonal growth in vitro, on poly-D-lysine-coated glass substrates, as diffusion in an effective external potential, representing the collective contribution of all causal influences on the growth cone. We use this approach to obtain effective growth rules that reveal an emergent regulatory mechanism for axonal pathfinding on these substrates.