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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 enables Netrin-1 attraction at low concentration. (A) Last brightfield images of typical growing axons. The growing neurite (yellow line), the initial orientation (dark dotted line), and the angle turned (rotating arrow) are shown. Bar, 30 μm. (B) Scatter plot of the angle turned versus the initial position (x). (C) Trajectory plots of growth cones in the different conditions, for initial positions between 300 and 700 μm. (D) The mean velocity (±SEM). n.s.: P > 0.05, Mann–Whitney test in which each condition is compared to the control. (E) 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.
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Fig3: Slit1 enables Netrin-1 attraction at low concentration. (A) Last brightfield images of typical growing axons. The growing neurite (yellow line), the initial orientation (dark dotted line), and the angle turned (rotating arrow) are shown. Bar, 30 μm. (B) Scatter plot of the angle turned versus the initial position (x). (C) Trajectory plots of growth cones in the different conditions, for initial positions between 300 and 700 μm. (D) The mean velocity (±SEM). n.s.: P > 0.05, Mann–Whitney test in which each condition is compared to the control. (E) 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.

Mentions: As it has been previously shown that moderate levels of Slit1 enables Netrin-1 attraction for rostral thalamic neurons [7], we characterized in vitro neuronal responses to a gradient of Netrin-1 with increasing uniform doses of Slit1. In the absence of Slit1, a steep 300 ng/ml Netrin-1 gradient did not induce significant attraction on rostral thalamic neurons (mean turning angle = 9° ± 8°, Figure 3), consistent with previous results obtained by co-culture experiments [7]. Combining the Netrin-1 gradient with a uniform concentration of Slit1 triggered significant attraction for rostral thalamic neurons, from 20 ng/ml concentration of Slit1 (mean turning angle = 29° ± 8°, Figure 3) and up to 40 ng/ml (mean turning angle = 27° ± 9°, Figure 3), indicating that a low level of Slit1 is sufficient to induce the response of rostral thalamic neurons to Netrin-1. Further increase in Slit1 concentration (100 ng/ml) did not elicit Netrin-1-induced attraction (mean turning angle = −6° ± 8°) (Figure 3F). In all the conditions tested, the mean velocity remained unchanged, showing that the effect induced by the combination of Netrin-1 and Slit1 is not a growth effect (Figure 3D). Our data indicates that moderate levels of Slit1 (from 20 to 40 ng/ml) are sufficient and necessary to induce Netrin-1 attraction. In contrast, no chemotactic effect is observed when Slit1 is applied as a gradient in the same range of concentrations (5 to 45 ng/ml for the 50 ng/ml Slit1 gradient, see Figure 2F). Combining Netrin-1 gradient with a uniform 100 ng/ml Slit1 concentration inhibits the response to the Netrin-1, suggesting that Slit1 enables Netrin-1 attraction only in a range of low concentration.Figure 3


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 enables Netrin-1 attraction at low concentration. (A) Last brightfield images of typical growing axons. The growing neurite (yellow line), the initial orientation (dark dotted line), and the angle turned (rotating arrow) are shown. Bar, 30 μm. (B) Scatter plot of the angle turned versus the initial position (x). (C) Trajectory plots of growth cones in the different conditions, for initial positions between 300 and 700 μm. (D) The mean velocity (±SEM). n.s.: P > 0.05, Mann–Whitney test in which each condition is compared to the control. (E) 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.
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Related In: Results  -  Collection

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Fig3: Slit1 enables Netrin-1 attraction at low concentration. (A) Last brightfield images of typical growing axons. The growing neurite (yellow line), the initial orientation (dark dotted line), and the angle turned (rotating arrow) are shown. Bar, 30 μm. (B) Scatter plot of the angle turned versus the initial position (x). (C) Trajectory plots of growth cones in the different conditions, for initial positions between 300 and 700 μm. (D) The mean velocity (±SEM). n.s.: P > 0.05, Mann–Whitney test in which each condition is compared to the control. (E) 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.
Mentions: As it has been previously shown that moderate levels of Slit1 enables Netrin-1 attraction for rostral thalamic neurons [7], we characterized in vitro neuronal responses to a gradient of Netrin-1 with increasing uniform doses of Slit1. In the absence of Slit1, a steep 300 ng/ml Netrin-1 gradient did not induce significant attraction on rostral thalamic neurons (mean turning angle = 9° ± 8°, Figure 3), consistent with previous results obtained by co-culture experiments [7]. Combining the Netrin-1 gradient with a uniform concentration of Slit1 triggered significant attraction for rostral thalamic neurons, from 20 ng/ml concentration of Slit1 (mean turning angle = 29° ± 8°, Figure 3) and up to 40 ng/ml (mean turning angle = 27° ± 9°, Figure 3), indicating that a low level of Slit1 is sufficient to induce the response of rostral thalamic neurons to Netrin-1. Further increase in Slit1 concentration (100 ng/ml) did not elicit Netrin-1-induced attraction (mean turning angle = −6° ± 8°) (Figure 3F). In all the conditions tested, the mean velocity remained unchanged, showing that the effect induced by the combination of Netrin-1 and Slit1 is not a growth effect (Figure 3D). Our data indicates that moderate levels of Slit1 (from 20 to 40 ng/ml) are sufficient and necessary to induce Netrin-1 attraction. In contrast, no chemotactic effect is observed when Slit1 is applied as a gradient in the same range of concentrations (5 to 45 ng/ml for the 50 ng/ml Slit1 gradient, see Figure 2F). Combining Netrin-1 gradient with a uniform 100 ng/ml Slit1 concentration inhibits the response to the Netrin-1, suggesting that Slit1 enables Netrin-1 attraction only in a range of low concentration.Figure 3

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