Guiding neuronal growth with light

Abstract

Control over neuronal growth is a fundamental objective in neuroscience, cell biology, developmental biology, biophysics, and biomedicine and is particularly important for the formation of neural circuits in vitro, as well as nerve regeneration in vivo [Zeck, G. & Fromherz, P. (2001) Proc. Natl. Acad. Sci. USA 98, 10457-10462]. We have shown experimentally that we can use weak optical forces to guide the direction taken by the leading edge, or growth cone, of a nerve cell. In actively extending growth cones, a laser spot is placed in front of a specific area of the nerve's leading edge, enhancing growth into the beam focus and resulting in guided neuronal turns as well as enhanced growth. The power of our laser is chosen so that the resulting gradient forces are sufficiently powerful to bias the actin polymerization-driven lamellipodia extension, but too weak to hold and move the growth cone. We are therefore using light to control a natural biological process, in sharp contrast to the established technique of optical tweezers [Ashkin, A. (1970) Phys. Rev. Lett. 24, 156-159; Ashkin, A. & Dziedzic, J. M. (1987) Science 235, 1517-1520], which uses large optical forces to manipulate entire structures. Our results therefore open an avenue to controlling neuronal growth in vitro and in vivo with a simple, noncontact technique.

Fig 1.

Experimental setup for the optical guidance of growing neurons. A laser spot (⊘ = 2–16 μm, power = 20–120 mW, λ = 800 nm) was placed with partial overlap in front of an actively extending growth cone. The overlap area was chosen in the direction of the preferred growth and to cover the actin cortex, which directly underlies the plasma membrane and drives the advancement of the leading edge of the nerve.

Fig 2.

Time sequences of optically guided turns of neurons and optically enhanced neuronal growth. Optically induced turns are shown for a time period of 40 min (Left) and 20 min (Right). The time interval between successive pictures is 10 min (Left) and 5 min (Right). The power of the laser spot is 100 mW (Left) and 60 mW (Right), and a red circle indicates the position of the laser spot (see Movies 1 and 2, which are published as supporting information on the PNAS web site, www.pnas.org). Optical control was achieved for extensive flat growth cones (Left) as well as for small, tube-like growth cones (Right). Before the laser altered the direction of the growth cone, the nerve was growing upward (Left) or to the right side (Right). The growth direction changes on the order of 90° under optical guidance. Note that the apparent change growth direction appears to be smaller because the axon straightens into the new direction.

Fig 3.

The parallel extension of two growth cones, one optically guided (right) and one normally growing, illustrates the increase in the speed of growth cone extension in the presence of a 20-mW laser spot (red circle) (see Movie 3, which is published as supporting information on the PNAS web site). The time interval between successive pictures is 10 min. The reference marks on the left are spaced in 5-μm steps.

Fig 4.

Superposition of the shapes of an actively extending growth cone under optical guidance. The same growth cone is shown in Fig. 2 Right. The progressing time is coded by the following series of colors: yellow (t = 0 min), red (t = 5 min), green (t = 10 min), and blue (t = 20 min). Two effects are clearly visible: stimulated by the laser, the small growth cone extends to the top of the picture in a pronounced lamellipodia structure with noticeable filopodia; the axonal stump, which does not actively participate in the growth process, changes its orientation by pointing more toward the top of the picture. Thus, the laser spot is able to induce significant changes in growth direction if the following axon can relax and avoid bends.

Fig 5.

Optically induced bifurcation of a growth cone. A growth cone, which is growing to the upper right, sprouts off an extension to the lower right under the influence of the beam marked by a circle. The last picture displays the distribution of actin filaments by rhodamine-phalloidin staining. Actin filaments are clearly accumulated at the areas of lamellipodia extension.