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Subrepellent doses of Slit1 promote Netrin-1 chemotactic responses in subsets of axons.

Dupin I, Lokmane L, Dahan M, Garel S, Studer V - Neural Dev (2015)

Bottom Line: In particular, it was recently found that the repellent Slit1 enables the attractive response of rostral thalamic axons to Netrin-1.We found that on rostral thalamic axons, only a subthreshold concentration of the repellent Slit1 triggered an attractive response to a gradient of Netrin-1.On hippocampal neurons, we similarly found that Slit1 alone is repulsive and a subthreshold concentration of Slit1 triggered a potent attractive or repulsive behavioral response to a gradient of Netrin-1, depending on the nature of the substrate.

View Article: PubMed Central - PubMed

Affiliation: University Bordeaux, IINS, UMR 5297, F-33000, Bordeaux, France. isabelle.dupin@u-bordeaux.fr.

ABSTRACT

Background: Axon pathfinding is controlled by guidance cues that elicit specific attractive or repulsive responses in growth cones. It has now become clear that some cues such as Netrin-1 can trigger either attraction or repulsion in a context-dependent manner. In particular, it was recently found that the repellent Slit1 enables the attractive response of rostral thalamic axons to Netrin-1. This finding raised the intriguing possibility that Netrin-1 and Slit1, two essential guidance cues, may act more generally in an unexpected combinatorial manner to orient specific axonal populations. To address this major issue, we have used an innovative microfluidic device compatible not only with dissociated neuronal cultures but also with explant cultures to systematically and quantitatively characterize the combinatorial activity of Slit1 and Netrin-1 on rostral thalamic axons as well as on hippocampal neurons.

Results: We found that on rostral thalamic axons, only a subthreshold concentration of the repellent Slit1 triggered an attractive response to a gradient of Netrin-1. On hippocampal neurons, we similarly found that Slit1 alone is repulsive and a subthreshold concentration of Slit1 triggered a potent attractive or repulsive behavioral response to a gradient of Netrin-1, depending on the nature of the substrate.

Conclusions: Our study reveals that at subthreshold repulsive levels, Slit1 acts as a potent promoter of both Netrin-1 attractive and repulsive activities on distinct neuronal cell types, thereby opening novel perspectives on the role of combinations of cues in brain wiring.

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Related in: MedlinePlus

Slit1 is repulsive for rostral thalamic axons at high concentration. (A) Brightfield image (left) of a typical experiment showing the explant cultured in a microwell on top of the microfluidic channels and the corresponding fluorescence image (right). (B) Example of a growing axon turning down a Slit1 gradient. (C) Defining the angle turned (β), the distance between initial/final position (d), and the initial position (x). The angle turned was defined as positive for turns towards the gradient and negative for turns away from the gradient. (D) The mean velocity (± standard deviation) defined as the distance ‘d’ divided by the time. n.s.: P > 0.05, Mann–Whitney test in which each condition is compared to the control. (E) Scatter plot of the angle turned versus the initial position (x). (F) The mean angle turned (β) (±SEM) for axons in the different conditions, for initial positions between 300 and 700 μm. Statistical differences are indicated *P < 0.05, Kruskal Wallis test with Dunn’s correction. (G) Trajectory plots of growth cones in the different conditions, for initial positions between 300 and 700 μm.
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Fig2: Slit1 is repulsive for rostral thalamic axons at high concentration. (A) Brightfield image (left) of a typical experiment showing the explant cultured in a microwell on top of the microfluidic channels and the corresponding fluorescence image (right). (B) Example of a growing axon turning down a Slit1 gradient. (C) Defining the angle turned (β), the distance between initial/final position (d), and the initial position (x). The angle turned was defined as positive for turns towards the gradient and negative for turns away from the gradient. (D) The mean velocity (± standard deviation) defined as the distance ‘d’ divided by the time. n.s.: P > 0.05, Mann–Whitney test in which each condition is compared to the control. (E) Scatter plot of the angle turned versus the initial position (x). (F) The mean angle turned (β) (±SEM) for axons in the different conditions, for initial positions between 300 and 700 μm. Statistical differences are indicated *P < 0.05, Kruskal Wallis test with Dunn’s correction. (G) Trajectory plots of growth cones in the different conditions, for initial positions between 300 and 700 μm.

Mentions: To investigate the combinatorial effects triggered by guidance cues, we used a microfluidic device similar to those described in [9] made of two fluidic channels separated by a gap of 200 μm (Figures 1A,B,C and 2A). One channel (the reservoir) is filled with a solution of guidance factors while the other contains only buffer. As a result, a gradient is generated in the cell chamber by diffusion through the porous track-etched membrane (Figure 1A,B,C). Using a tetramethylrhodamine-labeled 70 kDa dextran, we characterized by total internal reflection fluorescence microscopy (TIRFM) the concentration profile at the location of the neuronal culture. The gradient shape, which is dictated by the geometry of the system, is in good agreement with the diffusion-based model described in [10] (Figure 1C) and Additional file 1: Figure S1 and Additional file 2: Figure S2. In the central part of the chamber (300 to 700 μm area, Figure 1C), the concentration profile is approximately linear with a relative slope of 2%, defined as the change in concentration along the width of the growth cone (10 μm) divided by the concentration cR of factors in the reservoir. In our experiments, we only analyzed neurons located in this linear gradient area, and thus changing the concentration in the reservoir allowed us to simply change the gradient, characterized by the value of cR (in ng/mL). We also wish to stress that numerical simulations and experiments (Additional file 1: Figure S1 and Additional file 2: Figure S2) show that the concentration profile is not significantly perturbed by the presence of neurons or explants in the chamber.Figure 1


Subrepellent doses of Slit1 promote Netrin-1 chemotactic responses in subsets of axons.

Dupin I, Lokmane L, Dahan M, Garel S, Studer V - Neural Dev (2015)

Slit1 is repulsive for rostral thalamic axons at high concentration. (A) Brightfield image (left) of a typical experiment showing the explant cultured in a microwell on top of the microfluidic channels and the corresponding fluorescence image (right). (B) Example of a growing axon turning down a Slit1 gradient. (C) Defining the angle turned (β), the distance between initial/final position (d), and the initial position (x). The angle turned was defined as positive for turns towards the gradient and negative for turns away from the gradient. (D) The mean velocity (± standard deviation) defined as the distance ‘d’ divided by the time. n.s.: P > 0.05, Mann–Whitney test in which each condition is compared to the control. (E) Scatter plot of the angle turned versus the initial position (x). (F) The mean angle turned (β) (±SEM) for axons in the different conditions, for initial positions between 300 and 700 μm. Statistical differences are indicated *P < 0.05, Kruskal Wallis test with Dunn’s correction. (G) Trajectory plots of growth cones in the different conditions, for initial positions between 300 and 700 μm.
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Related In: Results  -  Collection

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Fig2: Slit1 is repulsive for rostral thalamic axons at high concentration. (A) Brightfield image (left) of a typical experiment showing the explant cultured in a microwell on top of the microfluidic channels and the corresponding fluorescence image (right). (B) Example of a growing axon turning down a Slit1 gradient. (C) Defining the angle turned (β), the distance between initial/final position (d), and the initial position (x). The angle turned was defined as positive for turns towards the gradient and negative for turns away from the gradient. (D) The mean velocity (± standard deviation) defined as the distance ‘d’ divided by the time. n.s.: P > 0.05, Mann–Whitney test in which each condition is compared to the control. (E) Scatter plot of the angle turned versus the initial position (x). (F) The mean angle turned (β) (±SEM) for axons in the different conditions, for initial positions between 300 and 700 μm. Statistical differences are indicated *P < 0.05, Kruskal Wallis test with Dunn’s correction. (G) Trajectory plots of growth cones in the different conditions, for initial positions between 300 and 700 μm.
Mentions: To investigate the combinatorial effects triggered by guidance cues, we used a microfluidic device similar to those described in [9] made of two fluidic channels separated by a gap of 200 μm (Figures 1A,B,C and 2A). One channel (the reservoir) is filled with a solution of guidance factors while the other contains only buffer. As a result, a gradient is generated in the cell chamber by diffusion through the porous track-etched membrane (Figure 1A,B,C). Using a tetramethylrhodamine-labeled 70 kDa dextran, we characterized by total internal reflection fluorescence microscopy (TIRFM) the concentration profile at the location of the neuronal culture. The gradient shape, which is dictated by the geometry of the system, is in good agreement with the diffusion-based model described in [10] (Figure 1C) and Additional file 1: Figure S1 and Additional file 2: Figure S2. In the central part of the chamber (300 to 700 μm area, Figure 1C), the concentration profile is approximately linear with a relative slope of 2%, defined as the change in concentration along the width of the growth cone (10 μm) divided by the concentration cR of factors in the reservoir. In our experiments, we only analyzed neurons located in this linear gradient area, and thus changing the concentration in the reservoir allowed us to simply change the gradient, characterized by the value of cR (in ng/mL). We also wish to stress that numerical simulations and experiments (Additional file 1: Figure S1 and Additional file 2: Figure S2) show that the concentration profile is not significantly perturbed by the presence of neurons or explants in the chamber.Figure 1

Bottom Line: In particular, it was recently found that the repellent Slit1 enables the attractive response of rostral thalamic axons to Netrin-1.We found that on rostral thalamic axons, only a subthreshold concentration of the repellent Slit1 triggered an attractive response to a gradient of Netrin-1.On hippocampal neurons, we similarly found that Slit1 alone is repulsive and a subthreshold concentration of Slit1 triggered a potent attractive or repulsive behavioral response to a gradient of Netrin-1, depending on the nature of the substrate.

View Article: PubMed Central - PubMed

Affiliation: University Bordeaux, IINS, UMR 5297, F-33000, Bordeaux, France. isabelle.dupin@u-bordeaux.fr.

ABSTRACT

Background: Axon pathfinding is controlled by guidance cues that elicit specific attractive or repulsive responses in growth cones. It has now become clear that some cues such as Netrin-1 can trigger either attraction or repulsion in a context-dependent manner. In particular, it was recently found that the repellent Slit1 enables the attractive response of rostral thalamic axons to Netrin-1. This finding raised the intriguing possibility that Netrin-1 and Slit1, two essential guidance cues, may act more generally in an unexpected combinatorial manner to orient specific axonal populations. To address this major issue, we have used an innovative microfluidic device compatible not only with dissociated neuronal cultures but also with explant cultures to systematically and quantitatively characterize the combinatorial activity of Slit1 and Netrin-1 on rostral thalamic axons as well as on hippocampal neurons.

Results: We found that on rostral thalamic axons, only a subthreshold concentration of the repellent Slit1 triggered an attractive response to a gradient of Netrin-1. On hippocampal neurons, we similarly found that Slit1 alone is repulsive and a subthreshold concentration of Slit1 triggered a potent attractive or repulsive behavioral response to a gradient of Netrin-1, depending on the nature of the substrate.

Conclusions: Our study reveals that at subthreshold repulsive levels, Slit1 acts as a potent promoter of both Netrin-1 attractive and repulsive activities on distinct neuronal cell types, thereby opening novel perspectives on the role of combinations of cues in brain wiring.

Show MeSH
Related in: MedlinePlus