Analysis of the growth cone turning assay for studying axon guidance

Pujic, Zac; Giacomantonio, Clare E.; Unni, Divya; Rosoff, William J.; Goodhill, Geoffrey J.

doi:10.1016/j.jneumeth.2008.01.014

Cited by 43 publications

(42 citation statements)

References 18 publications

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“…The pipette was loaded with either recombinant mouse Netrin1 (10 µg/mL; R&D Systems) or PBS and mixed with 70-kDa dextran labeled with fluorescent tetramethylrhodamine (Molecular Probes Inc.) to monitor the gradient produced. Steep gradients with a 10-15% change in concentration across 10 µm were generated using the pulsatile ejection method (Pujic et al 2008). Images of the growing axon were taken using the 20× objective of a Zeiss Axio Observer inverted microscope and AxioVision 4 software at 60 s intervals for 1 h. The trace of each axon, its turning angle, and the distance of growth were calculated as described in Thompson et al (2011).…”

Section: Growth Cone Turning Assaymentioning

confidence: 99%

Netrin-DCC Signaling Regulates Corpus Callosum Formation Through Attraction of Pioneering Axons and by Modulating Slit2-Mediated Repulsion

et al. 2013

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The left and right sides of the nervous system communicate via commissural axons that cross the midline during development using evolutionarily conserved molecules. These guidance cues have been particularly well studied in the mammalian spinal cord, but it remains unclear whether these guidance mechanisms for commissural axons are similar in the developing forebrain, in particular for the corpus callosum, the largest and most important commissure for cortical function. Here, we show that Netrin1 initially attracts callosal pioneering axons derived from the cingulate cortex, but surprisingly is not attractive for the neocortical callosal axons that make up the bulk of the projection. Instead, we show that Netrindeleted in colorectal cancer signaling acts in a fundamentally different manner, to prevent the Slit2-mediated repulsion of precrossing axons thereby allowing them to approach and cross the midline. These results provide the first evidence for how callosal axons integrate multiple guidance cues to navigate the midline.

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Section: Growth Cone Turning Assaymentioning

confidence: 99%

Netrin-DCC Signaling Regulates Corpus Callosum Formation Through Attraction of Pioneering Axons and by Modulating Slit2-Mediated Repulsion

et al. 2013

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“…Micropipette turning assays have commonly been used for studying neurite guidance in vitro 12 . Here, the micropipette serves as a continuous point source of chemoattractant, producing a growth factor gradient by diffusion to guide neurite growth 13,14 .…”

Section: Page 8 Of 49 Lab On a Chip -For Review Onlymentioning

confidence: 99%

A high-throughput microfluidic assay to study neurite response to growth factor gradients

et al. 2011

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Growth factor gradients have been implicated in many biological phenomena, including cell migration, neurite outgrowth and guidance in the nervous system. Current in vitro assays to study the effect of growth factors on neurite guidance are limited by their suitability for only substrate-bound gradients and 2D cultures. Here we describe a novel three-channel microfluidic device to study the role of chemogradients on neurite outgrowth and guidance in 3D scaffolds. Neurons packed in the bottom channel onto the collagen gel surface extended their neurites into the gel in three dimensions, and the growing neurites were exposed to a chemogradient orthogonal to the direction of their growth to quantify their response and turning. Experimental and computational studies showed that a linear stable chemogradient could be established in these devices within 30 min, and lasting for up to 48 h. Cell culture studies revealed the dramatic effect of chemoattractive (netrin-1, brain pulp) and chemorepulsive (slit-2) gradients on the migration and neurite guidance of hippocampal and dorsal root ganglion neurons cultured in these devices. The stable chemogradients in these 3-channel devices could not only be used to screen potential drugs suitable for neuron pathway regeneration under disease/ injury conditions, but also to study cancer cell migration and cell-cell interactions. AbstractStudying neurite guidance by diffusible or substrate bound gradients is challenging with current techniques. In this study, we present the design, fabrication and utility of a microfluidic device to study neurite guidance under chemogradients. Experimental and computational studies demonstrated the establishment of a steep gradient of guidance cue within 30 min and stable for up to 48 h. The gradient was found to be insensitive to external perturbations such as media change and movement of device. The effects of netrin-1 (0.1-10 µg/mL) and brain pulp (0.1 µL/mL) were evaluated for their chemoattractive potential on neurite turning, while slit-2 (62.5 or 250 ng/mL) was studied for its chemorepellant properties. Hippocampal or dorsal root ganglion (DRG) neurons were seeded into a micro-channel and packed onto the surface of a 3D collagen gel. Neurites grew into the matrix in three dimensions, and a gradient of guidance cue was created orthogonal to the direction of neurite growth to impact guidance. The average turning angle of each neurite was measured and averaged across multiple devices cultured under similar conditions to quantify the effect of guidance cue gradient. Significant positive turning towards gradient were measured in the presence of brain pulp and netrin-1 (1 µg/mL), relative to control cultures which received no external guidance cue (p < 0.001). Netrin-1 released from transfected fibroblasts had the most positive turning effect of all the chemoattractive cues tested (p

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“…To do this we utilized the well-established growth cone turning assay on dissociated SCG cells. This assay produces gradients with steepness of roughly a 10 -15% change in concentration across 10 µm, and is commonly used to assess growth cone turning over about one hour of growth (Lohof et al, 1992;Pujic et al, 2008). When we used a concentration of 10 µM NGF in the pipette, growth cones from dissociated SCG neurons showed significant attraction up the gradient (mean turning angle = 14.6 ± 4.7°) compared to the control condition (mean turning angle = 0.02 ± 2.7°; p = 0.02, Kolmogorov-Smirnov (KS) test, Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Growth cones with a straight trailing axon of more than 20 µm were selected for the assay. Steep gradients of 10 -15% change in concentration across 10 µm were generated using the pulsatile ejection method reported previously by Lohof et al (1992) (see also Pujic et al, 2008). 70 kDa dextran labeled 6 with fluorescent tetramethylrhodamine (Molecular Probes Inc., Melbourne, Australia) was added to the pipette solution to monitor the chemical gradient produced.…”

Section: Methodsmentioning

confidence: 99%

Cyclic nucleotide-dependent switching of mammalian axon guidance depends on gradient steepness

Thompson

Pujic

Richards

et al. 2011

Molecular and Cellular Neuroscience

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Correct wiring of the nervous system during development requires axons to respond appropriately to gradients of attractive and repulsive guidance cues. However, the steepness and concentration of these gradients vary in vivo, for instance, with distance from the target. Understanding how these changing conditions affect the navigation strategies used by developing axons is important for understanding how they are guided over long distances. Previous work has shown that cyclic nucleotide levels determine whether axons are attracted or repelled by steep gradients of the same guidance cue, but it is unknown whether this is also true for shallow gradients. We therefore investigated the guidance responses of rat superior cervical ganglion (SCG) axons in both steep and shallow gradients of nerve growth factor (NGF). In steep gradients we found that cyclic nucleotide-dependent switching occurred, consistent with previous reports. Surprisingly however, we found that in shallow NGF gradients, cyclic nucleotide-dependent switching did not occur. These results suggest that there may be substantial differences in the way axons respond to gradient-based guidance cues depending on where they are within the gradient.2

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Analysis of the growth cone turning assay for studying axon guidance

Cited by 43 publications

References 18 publications

Netrin-DCC Signaling Regulates Corpus Callosum Formation Through Attraction of Pioneering Axons and by Modulating Slit2-Mediated Repulsion

Netrin-DCC Signaling Regulates Corpus Callosum Formation Through Attraction of Pioneering Axons and by Modulating Slit2-Mediated Repulsion

A high-throughput microfluidic assay to study neurite response to growth factor gradients

Cyclic nucleotide-dependent switching of mammalian axon guidance depends on gradient steepness

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