Limits...
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.

Show MeSH

Related in: MedlinePlus

Slit1 triggers Netrin-1 repulsion for hippocampal neurons on PLL-coated substrate. (A) Last brighfield images of typical growing axons on PLL-coated microwells. The growing neurite (yellow line), the initial orientation (dark dotted line), and the angle turned (rotating arrow) are shown. Bar, 20 μm. (B) Scatter plot of the angle turned versus the initial position (x). (C) The mean velocity (±SEM). n.s.: P > 0.05, Mann–Whitney test in which each condition is compared to the control. (D) 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. (E) Schema showing the responses of hippocampal neurons to different stimulations depending on the nature of substrate coating. On PLL and PLL-laminin-coated microwells, hippocampal neurons are repelled by a Slit1 gradient at high concentration. When submitted to the combination of a Netrin-1 gradient with a low uniform concentration of Slit1, the response of hippocampal growth cones is shifted from repulsion on PLL-coated substrate to an attraction on PLL-laminin-coated substrates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4373007&req=5

Fig5: Slit1 triggers Netrin-1 repulsion for hippocampal neurons on PLL-coated substrate. (A) Last brighfield images of typical growing axons on PLL-coated microwells. The growing neurite (yellow line), the initial orientation (dark dotted line), and the angle turned (rotating arrow) are shown. Bar, 20 μm. (B) Scatter plot of the angle turned versus the initial position (x). (C) The mean velocity (±SEM). n.s.: P > 0.05, Mann–Whitney test in which each condition is compared to the control. (D) 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. (E) Schema showing the responses of hippocampal neurons to different stimulations depending on the nature of substrate coating. On PLL and PLL-laminin-coated microwells, hippocampal neurons are repelled by a Slit1 gradient at high concentration. When submitted to the combination of a Netrin-1 gradient with a low uniform concentration of Slit1, the response of hippocampal growth cones is shifted from repulsion on PLL-coated substrate to an attraction on PLL-laminin-coated substrates.

Mentions: We performed in parallel the same experiments with hippocampal neurons growing on a PLL-coated substrate, which is the classical culture condition for these neurons. Similarly, on PLL or on a PLL-laminin substrate, a repulsion can be observed for hippocampal neurons submitted to a 200 ng/ml Slit1 gradient (mean turning angle = −22° ± 11° on PLL substrate) while a 300 ng/ml Netrin-1 gradient did not trigger significant chemotactic activity (mean turning angle = 10° ± 12° on PLL substrate) (Figure 5A,B,D). Strikingly, the response to the combination of both cues was shifted from attraction to repulsion when hippocampal neurons were growing on PLL substrate (mean turning angle = −22° ± 11°, Figure 5A,B,D) suggesting that the extracellular matrix can regulate the response of growth cones to diffusible cues. This shift was specific to the combination Netrin-1/Slit1 since Slit1 alone elicited a repulsive response in hippocampal axons on either PLL or PLL-laminin-coated substrate. In all the conditions, the axons grew at similar velocities (Figures 4C and 5C). Altogether, our results show that Slit1 enables the chemotactic activity of Netrin-1 in hippocampal neurons, irrespective of whether this activity is attractive or repulsive (Figure 5E).Figure 5


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 triggers Netrin-1 repulsion for hippocampal neurons on PLL-coated substrate. (A) Last brighfield images of typical growing axons on PLL-coated microwells. The growing neurite (yellow line), the initial orientation (dark dotted line), and the angle turned (rotating arrow) are shown. Bar, 20 μm. (B) Scatter plot of the angle turned versus the initial position (x). (C) The mean velocity (±SEM). n.s.: P > 0.05, Mann–Whitney test in which each condition is compared to the control. (D) 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. (E) Schema showing the responses of hippocampal neurons to different stimulations depending on the nature of substrate coating. On PLL and PLL-laminin-coated microwells, hippocampal neurons are repelled by a Slit1 gradient at high concentration. When submitted to the combination of a Netrin-1 gradient with a low uniform concentration of Slit1, the response of hippocampal growth cones is shifted from repulsion on PLL-coated substrate to an attraction on PLL-laminin-coated substrates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4373007&req=5

Fig5: Slit1 triggers Netrin-1 repulsion for hippocampal neurons on PLL-coated substrate. (A) Last brighfield images of typical growing axons on PLL-coated microwells. The growing neurite (yellow line), the initial orientation (dark dotted line), and the angle turned (rotating arrow) are shown. Bar, 20 μm. (B) Scatter plot of the angle turned versus the initial position (x). (C) The mean velocity (±SEM). n.s.: P > 0.05, Mann–Whitney test in which each condition is compared to the control. (D) 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. (E) Schema showing the responses of hippocampal neurons to different stimulations depending on the nature of substrate coating. On PLL and PLL-laminin-coated microwells, hippocampal neurons are repelled by a Slit1 gradient at high concentration. When submitted to the combination of a Netrin-1 gradient with a low uniform concentration of Slit1, the response of hippocampal growth cones is shifted from repulsion on PLL-coated substrate to an attraction on PLL-laminin-coated substrates.
Mentions: We performed in parallel the same experiments with hippocampal neurons growing on a PLL-coated substrate, which is the classical culture condition for these neurons. Similarly, on PLL or on a PLL-laminin substrate, a repulsion can be observed for hippocampal neurons submitted to a 200 ng/ml Slit1 gradient (mean turning angle = −22° ± 11° on PLL substrate) while a 300 ng/ml Netrin-1 gradient did not trigger significant chemotactic activity (mean turning angle = 10° ± 12° on PLL substrate) (Figure 5A,B,D). Strikingly, the response to the combination of both cues was shifted from attraction to repulsion when hippocampal neurons were growing on PLL substrate (mean turning angle = −22° ± 11°, Figure 5A,B,D) suggesting that the extracellular matrix can regulate the response of growth cones to diffusible cues. This shift was specific to the combination Netrin-1/Slit1 since Slit1 alone elicited a repulsive response in hippocampal axons on either PLL or PLL-laminin-coated substrate. In all the conditions, the axons grew at similar velocities (Figures 4C and 5C). Altogether, our results show that Slit1 enables the chemotactic activity of Netrin-1 in hippocampal neurons, irrespective of whether this activity is attractive or repulsive (Figure 5E).Figure 5

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