Neurite branch retraction is caused by a threshold-dependent mechanical impact

Abstract

Recent results indicate that, in addition to chemical cues, mechanical stimuli may also impact neuronal growth. For instance, unlike most other cell types, neurons prefer soft substrates. However, the mechanisms responsible for the neuronal affinity for soft substrates have not yet been identified. In this study, we show that, in vitro, neurons continuously probe their mechanical environment. Growth cones visibly deform substrates with a compliance commensurate with their own. To understand the sensing of stiff substrates by growth cones, we investigated their precise temporal response to well-defined mechanical stress. When the applied stress exceeded a threshold of 274 +/- 41 pN/microm(2), neurons retracted and re-extended their processes, thereby enabling exploration of alternative directions. A calcium influx through stretch-activated ion channels and the detachment of adhesion sites were prerequisites for this retraction. Our data illustrate how growing neurons may detect and avoid stiff substrates--as a mechanism involved in axonal branch pruning--and provide what we believe is novel support of the idea that mechanics may act as guidance cue for neuronal growth.

Figure 1

Deformation map of a growth cone plated on a 200 Pa polyacrylamide gel. The color coding gives the deformation of the gel; the arrows indicate the direction of deformation. The growth cone, whose edge is indicated by the black line, was able to deform the gel up to a maximum of 600 nm, and at substrate stiffness exceeding 400 Pa the deformations drop below optical resolution. Scale bar = 10 μm.

Figure 2

Response of NG108-15 processes to mechanical stimulation of their growth cone. (A) Phase contrast image of a neuronal growth cone. (B) After mechanical stress application, the growth cone collapsed. Scale bar = 10 μm, also applies for A. (C–F) Neurite retraction after mechanical stimulation. (C) NG108-15 cell, neurite is growing toward the left upper corner of the image. (D) When mechanical stress was applied to the leading edge of its growth cone (arrow: SFM cantilever), the growth cone collapsed and the neurite retracted (E). (F) Eventually, the neuronal process grew in a new direction (dashed line: initial growth direction). Scale bar = 30 μm, also applies for C–E.

Figure 3

Response of PC12 processes to mechanical stimulation of their growth cone (cf. Movie S2). (A) Phase contrast image of a neuronal growth cone. (B) After application of mechanical stress, the growth cone collapsed. Scale bar = 10 μm, also applies for A. (C–G) Neurite retraction after mechanical stimulation. (C) A neurite is growing toward the left upper corner of the image. (D) When a mechanical suprathreshold stimulus (>274 pN/μm²) was applied to the leading edge of its growth cone (arrow: SFM cantilever), the growth cone collapsed and the neurite retracted and assumed a coil-like shape (arrow in E). (F) Subsequently, a new growth cone established, tension recovered, and the neurite straightened again. (G) Finally, the neuronal process grew in a new direction; the dashed line indicates the initial direction of neurite growth. Scale bar = 50 μm, also applies for C–F.

Figure 4

[Ca²⁺]_i within a mechanically stimulated neuron. (A) [Ca²⁺]_i before, (B) during, and (C) 5 s, (D) 15 s, and (E) 30 s after mechanical stimulation of the growth cone (arrow in A; arrow head: neurite; asterisk: soma). The increase in [Ca²⁺]_i spread from the area of mechanical contact into the neurite and in neighboring structures such as side branches if applicable (D,E). The color represents [Ca²⁺]_i. Color coding and scale bar (50 μm) in E also apply for A–D.

Figure 5

Relative change in neurite length 300 s after mechanical suprathreshold stimulation. In normal medium, neurites retracted to 56 ± 10% of their original length (n = 12, mean ± SE). When 25 μM gadolinium chloride, which is a blocker of SACs (44), was applied to the solution, no significant retraction of the neurites could be triggered (n = 9, mean ± SE, p < 0.01).

Figure 6

Interference reflection microscopy images of a neuronal growth cone. Structures that are in close contact to the substrate appear dark. (A) Growth cone before stress application. Adhesion sites, which are the dark structures visible, were densely packed at the growth cone's leading edge (arrow). The arrowhead points toward the direction of neurite extension. (B) Neuronal process 3 s after mechanical stimulation. The adhesion sites of the growth cone with the substrate disappeared concomitantly with increased [Ca²⁺]_i. Scale bar = 25 μm, also applies for A.

Figure 7

Scheme summarizing the effects of mechanical cues on neurite growth. (A) Growth cones are attached to the substrate by focal adhesions (FAs), which are connected to contractile elements (CE), and that may be linked to SACs. Neurons exert contractile forces (arrows) on their environment via FAs. These forces act on both focal adhesions and SACs. (B) If the substrate stiffness (E_s) is below the critical threshold, which corresponds to the growth cone's own compliance (E_c), the substrate is deformed and the channels remain closed. (C) When the substrate stiffness exceeds that of the growth cone, the force exerted on the SACs may be sufficient to trigger their opening, leading to a calcium influx from the extracellular space. (D) Illustration of neuronal growth in dependence on substrate compliance. Left: If the neuron grows on a soft substrate, the growth cone advances unhindered. Right: If the neuron approaches a hard substrate, more and more force generated by the contractile elements is transmitted to the SACs, until they open and calcium enters the interior of the cell, triggering growth cone collapse and neurite retraction by increasing its contractility and/or by destabilizing its focal adhesions. Subsequently, the growth cone adheres again to the substrate and grows into a new direction. This mechanism may slow down the effective neuronal growth rate in regions that are stiffer than the threshold, and it might ultimately be used as guidance cue during neuronal pathfinding.