Optogenetic oligomerization of Rab GTPases regulates intracellular membrane trafficking.

Intracellular membrane trafficking, which is involved in diverse cellular processes, is dynamic and difficult to study in a spatiotemporal manner. Here we report an optogenetic strategy, termed light-activated reversible inhibition by assembled trap of intracellular membranes (IM-LARIAT), that uses various Rab GTPases combined with blue-light-induced hetero-interaction between cryptochrome 2 and CIB1. In this system, illumination induces a rapid and reversible intracellular membrane aggregation that disrupts the dynamics and functions of the targeted membrane. We applied IM-LARIAT to specifically perturb several Rab-mediated trafficking processes, including receptor transport, protein sorting and secretion, and signaling initiated from endosomes. We finally used this tool to reveal different functions of local Rab5-mediated and Rab11-mediated membrane trafficking in growth cones and soma of young hippocampal neurons. Our results show that IM-LARIAT is a versatile tool that can be used to dissect spatiotemporal functions of intracellular membranes in diverse systems.

Optogenetic control of PIP3: PIP3 is sufficient to induce the actin-based active part of growth cones and is regulated via endocytosis.

Phosphatidylinositol-3,4,5-trisphosphate (PIP3) is highly regulated in a spatiotemporal manner and plays multiple roles in individual cells. However, the local dynamics and primary functions of PIP3 in developing neurons remain unclear because of a lack of techniques for manipulating PIP3 spatiotemporally. We addressed this issue by combining optogenetic control and observation of endogenous PIP3 signaling. Endogenous PIP3 was abundant in actin-rich structures such as growth cones and "waves", and PIP3-rich plasma membranes moved actively within growth cones. To study the role of PIP3 in developing neurons, we developed a PI3K photoswitch that can induce production of PIP3 at specific locations upon blue light exposure. We succeeded in producing PIP3 locally in mouse hippocampal neurons. Local PIP3 elevation at neurite tips did not induce neurite elongation, but it was sufficient to induce the formation of filopodia and lamellipodia. Interestingly, ectopic PIP3 elevation alone activated membranes to form actin-based structures whose behavior was similar to that of growth-cone-like "waves". We also found that endocytosis regulates effective PIP3 concentration at plasma membranes. These results revealed the local dynamics and primary functions of PIP3, providing fundamental information about PIP3 signaling in neurons.

Analysis of laser-induced heating in optical neuronal guidance.

Recently, it has been shown that it is possible to control the growth direction of neuronal growth cones by stimulation with weak laser light; an effect dubbed optical neuronal guidance. The effect exists for a broad range of laser wavelengths, spot sizes, spot intensities, optical intensity profiles and beam modulations, but it is unknown which biophysical mechanisms govern it. Based on thermodynamic modeling and simulation using published experimental parameters as input, we argue that the guidance is linked to heating. Until now, temperature effects due to laser-induced heating of the guided neuron have been neglected in the optical neuronal guidance literature. The results of our finite-element-method simulations show the relevance of the temperature field in optical guidance experiments and are consistent with published experimental results and modeling in the field of optical traps. Furthermore, we propose two experiments designed to test this hypotheses experimentally. For one of these experiments, we have designed a microfluidic platform, to be made using standard microfabrication techniques, for incubation of neurons in temperature gradients on micrometer lengthscales.

Signalling effect of NIR pulsed lasers on axonal growth.

In this work we show that a pulsed laser light placed at a distance is able to modulate the growth of axons of primary neuronal cell cultures. In our experiments continuous wave (CW), chopped CW and modelocked fs (FS) laser light was focused through a microscope objective to a point placed at a distance of about 15 microm from the growth cone. We found that CW light does not produce any significant influence on the axon growth. In contrast, when using pulsed light (chopped CW light or FS pulses), the beam was able to modify the trajectory of the axons, attracting approximately 45% of the observed cases to the beam spot. Such effect could possibly indicate the capacity of neurons to interpret the pulsating NIR light as the source of other nearby cells, resulting in extension of processes in the direction of the source.

Optical neuronal guidance in three-dimensional matrices.

We demonstrate effective guidance of neurites extending from PC12 cells in a three-dimensional collagen matrix using a focused infrared laser. Processes can be redirected in an arbitrarily chosen direction in the imaging plane in approximately 30 min with an 80% success rate. In addition, the application of the laser beam significantly increases the rate of neurite outgrowth. These results extend previous observations on 2D coated glass coverslips. We find that the morphology of growth cones is very different in 3D than in 2D, and that this difference suggests that the filopodia play a key role in optical guidance. This powerful, flexible, non-contact guidance technique has potentially broad applications in tissues and engineered environments.

Growth cone collapse and neurite retractions: an approach to examine X-irradiation affects on neuron cells.

The growth cone is a structure at the terminal of a neurite that plays an important role in the growth of the neurite. The growth cone collapse assay is considered to be a useful method to quantify the effects of various factors on nerve tissue. Here, we investigated the effect of x-irradiation on growth cones and neurites and also the comparative radiosensitivity of different neurons. Dorsal root ganglia and sympathetic chain ganglion were isolated from day-8 and -16 chick embryos and cultured for 20 h. Neurons were then exposed to x-irradiation and morphological changes were quantitatively evaluated by growth cone collapse assay. Cell viability was examined using TUNEL and WST-1 assays. The results showed that radiation induced growth cone collapse and neurite retraction in a time- and exposure-responsive manner. Growth cone collapse, apoptosis and WST-1 assays showed that no significant difference between the neurons throughout the study period (p > or = 0.5) after irradiation. Both types of day-8 neurons were more radio-sensitive than day-16 neurons (p < or = 0.05). The time course of the growth cone collapse was significantly correlated with the apoptotic and cell viability responses at different irradiation doses. Growth cone collapse may represent a useful marker for assaying the effect of x-irradiation on normal cell neurons.

Electric field effects on human spinal injury: Is there a basis in the in vitro studies?

An important basis for the clinical application of small DC electric current to mammalian spinal injury is the responses of neurons in culture to applied electric fields. Our recent finding that zebrafish neurons were unresponsive to applied fields prompted us to critically examine previous results. We conclude that compelling evidence for neuronal guidance and directional stimulation of growth toward either the cathode or anode in an electric field exists only for cultured Xenopus neurons, and not for any mammalian neurons. No basis for the reported success in treating spinal injury exists in the in vitro studies, and considerable research will be required if the conditions of field application in mammalian spinal injury are to be optimized.

Adenomatous polyposis coli is differentially distributed in growth cones and modulates their steering.

Axonal steering reactions depend on the transformation of environmental information into internal, directed structures, which is achieved by differential modulation of the growth cone cytoskeleton; key elements are the microtubules, which are regulated in their dynamics by microtubule-associated proteins (MAPs). We investigated a potential role of the MAP adenomatous polyposis coli (APC) for growing axons, employing embryonic visual system as a model system. APC is concentrated in the distalmost (i.e., growing) region of retinal ganglion cell axons in vivo and in vitro. Within the growth cone, APC is enriched in the central domain; it only partially colocalizes with microtubules. When axons are induced to turn toward a cell or away from a substrate border, APC is present in the protruding and absent from the collapsing growth cone regions, thus indicating the future growth direction of the axon. To assess the functional role of the differential distribution of APC in navigating growth cones, the protein was inactivated via micro-scale chromophore-assisted laser inactivation in one half of the growth cone. If the N-terminal APC region (crucial for its oligomerization) is locally inactivated, the treated growth cone side collapses and the axon turns away. In contrast, if the 20 aa repeats in the middle region of APC (which can negatively regulate its microtubule association) are inactivated, protrusions are formed and the growth cone turns toward. Our data thus demonstrate a crucial role of APC for axon steering attributable to its multifunctional domain structure and differential distribution in the growth cone.

Fast regulation of axonal growth cone motility by electrical activity.

Axonal growth cones are responsible for the correct guidance of developing axons and the establishment of functional neural networks. They are highly motile because of fast and continuous rearrangements of their actin-rich cytoskeleton. Here we have used live imaging of axonal growth cones of hippocampal neurons in culture and quantified their motility with a temporal resolution of 2 s. Using novel methods of analysis of growth cone dynamics, we show that transient activation of kainate receptors by bath-applied kainate induced a fast and reversible growth cone stalling. This effect depends on electrical activity and can be mimicked by the transient discharge of action potentials elicited in the neuron by intracellular current injections at the somatic level through a patch pipette. Growth cone stalling induced by electrical stimulation is mediated by calcium entry from the extracellular medium as well as by calcium release from intracellular stores that define spatially restricted microdomains directly affecting cytoskeletal dynamics. We propose that growth cone motility is dynamically controlled by transient bursts of spontaneous electrical activity, which constitutes a prominent feature of developing neural networks in vivo.

Electroporation of neurons and growth cones in Aplysia californica.

Specific labeling of individual neurons and neuronal processes is virtually an everyday task for neuroscientists. Many traditional ways for delivery of intracellular dyes have limitations in terms of speed, efficiency and reproducibility. Electroporation is a fast, reliable and efficient method to deliver microscopic amounts of polar and charged molecules into neurons and their compartments such as individual neurites and growth cones. Here, we present a simple and highly effective procedure for intracellular labeling of individual Aplysia neurons both in intact ganglia and in cell culture. Pleural mechanoreceptor neurons have been used as illustrative examples to demonstrate applicability of direct and local labeling of the smallest individual neurites (< 2 microm) and single growth cones. Specifically, a 3-s train of 1.0 V hyperpolarizing pulses at 50 Hz effectively filled discrete neurites in contact with the tip of the micropipette with no dye transfer visible to other, non-contacted neurites. Application of this localized dye labeling technique to single neurites reveals a surprisingly complex morphology for patterns of axonal branching in culture. The protocol can be easily applied to a variety of models in neuroscience including accessible nervous systems of invertebrate animals.

Inactivation of myelin-associated glycoprotein enhances optic nerve regeneration.

CNS regeneration in higher vertebrates is a long sought after goal in neuroscience. The lack of regeneration is attributable in part to inhibitory factors found in myelin (Caroni and Schwab, 1988a). Myelin-associated glycoprotein (MAG) is an abundant myelin protein that inhibits neurite outgrowth in vitro (McKerracher et al., 1994; Mukhopadhyay et al., 1994), but its role in regeneration remains controversial. To address this role, we performed nerve crush on embryonic day 15 chick retina-optic nerve explants and then acutely eliminated MAG function along the nerve using chromophore-assisted laser inactivation (CALI). CALI of MAG permitted significant regrowth of retinal axons past the site of lesion containing CNS myelin in contrast to various control treatments. Electron microscopy of the site of nerve crush shows abundant regenerating axons crossing the gap. When crushed optic nerve was retrogradely labeled at the nerve stump, no labeling of retinal neurons was observed. In contrast, labeling of CALI of MAG-treated crushed optic nerve showed significant retinal labeling (89 +/- 16 cells per square millimeter), a value indistinguishable from that seen with non-crushed nerve (98 +/- 13 cells per square millimeter). These findings implicate MAG as an important component of the myelin-derived inhibition of nerve regeneration. The acute loss of MAG function can promote significant axon growth across a site of CNS nerve damage.

Guiding neuronal growth with light.

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.