Limits...
Defective microglial development in the hippocampus of Cx3cr1 deficient mice.

Pagani F, Paolicelli RC, Murana E, Cortese B, Di Angelantonio S, Zurolo E, Guiducci E, Ferreira TA, Garofalo S, Catalano M, D'Alessandro G, Porzia A, Peruzzi G, Mainiero F, Limatola C, Gross CT, Ragozzino D - Front Cell Neurosci (2015)

Bottom Line: We found that fractalkine signaling is necessary for the development of several morphological and physiological features of microglia.Fractalkine signaling also influenced microglial morphology and ability to extend processes in response to ATP following its focal application to the slice.Our results reveal the developmental profile of several morphological and physiological properties of microglia and demonstrate that these processes are modulated by fractalkine signaling.

View Article: PubMed Central - PubMed

Affiliation: Center for Life Nanoscience - Istituto Italiano di Tecnologia@Sapienza, Rome Italy.

ABSTRACT
Microglial cells participate in brain development and influence neuronal loss and synaptic maturation. Fractalkine is an important neuronal chemokine whose expression increases during development and that can influence microglia function via the fractalkine receptor, CX3CR1. Mice lacking Cx3cr1 show a variety of neuronal defects thought to be the result of deficient microglia function. Activation of CX3CR1 is important for the proper migration of microglia to sites of injury and into the brain during development. However, little is known about how fractalkine modulates microglial properties during development. Here we examined microglial morphology, response to ATP, and K(+) current properties in acute brain slices from Cx3cr1 knockout mice across postnatal hippocampal development. We found that fractalkine signaling is necessary for the development of several morphological and physiological features of microglia. Specifically, we found that the occurrence of an outward rectifying K(+) current, typical of activated microglia, that peaked during the second and third postnatal week, was reduced in Cx3cr1 knockout mice. Fractalkine signaling also influenced microglial morphology and ability to extend processes in response to ATP following its focal application to the slice. Our results reveal the developmental profile of several morphological and physiological properties of microglia and demonstrate that these processes are modulated by fractalkine signaling.

No MeSH data available.


Related in: MedlinePlus

Tracking analysis of single processes in CX3CR1+/GFP and CX3CR1GFP/GFP microglia. (A) Left, plot of spatial x-y coordinates respect to the ATP pipette tip of microglial processes in CX3CR1+/GFP (purple symbols, n = 133 processes/8 fields) and CX3CR1GFP/GFP hippocampal slices (orange symbols, n = 89/8) at start of recordings (t0). Right, cumulative distributions of radial distances of microglial processes from the pipette tip in CX3CR1+/GFP (purple) or CX3CR1GFP/GFP (orange) slices, at t0. Note that CX3CR1GFP/GFP processes are significantly more distant than CX3CR1+/GFP ones (∗p < 0.05, Kolmogorov-Smirnov test). (B) Left, plot of spatial x-y coordinates of CX3CR1+/GFP (purple symbols, n = 209/8) and CX3CR1GFP/GFP (orange symbols, n = 161/8) microglial processes 45 min after ATP application (t45). Right, cumulative distributions of radial distances of microglial processes in CX3CR1+/GFP (purple) or CX3CR1GFP/GFP (orange) slices, at t45. Note that at t45 the majority of CX3CR1GFP/GFP processes do not reach the ATP pipette (∗∗p < 0.0001, Kolmogorov-Smirnov test). (C) Box charts of displacement (left) and directionality (right) of CX3CR1+/GFP (purple) and CX3CR1GFP/GFP (orange) microglial processes. In slices from Cx3cr1+/GFP mice displacement and directionality are significantly higher than in those from Cx3cr1GFP/GFP mice (∗∗∗p < 0.0001, t-test), (D) Correlation plot showing mean instantaneous velocities vs. radial distance of CX3CR1+/GFP (purple) and CX3CR1GFP/GFP (orange) microglial processes. The velocity of CX3CR1+/GFP processes increase significantly 7 mm far from the ATP pipette tip (∗p < 0.001, Kruskal-Wallis One Way ANOVA on Ranks, and p < 0.05 multiple comparison Dunn’s method versus 15, 25, and 35 μm radial distance). Note that in the proximity of the ATP pipette, the velocity of CX3CR1GFP/GFP processes is significantly slower than that of CX3CR1+/GFP processes (#p < 0.01 t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4379915&req=5

Figure 4: Tracking analysis of single processes in CX3CR1+/GFP and CX3CR1GFP/GFP microglia. (A) Left, plot of spatial x-y coordinates respect to the ATP pipette tip of microglial processes in CX3CR1+/GFP (purple symbols, n = 133 processes/8 fields) and CX3CR1GFP/GFP hippocampal slices (orange symbols, n = 89/8) at start of recordings (t0). Right, cumulative distributions of radial distances of microglial processes from the pipette tip in CX3CR1+/GFP (purple) or CX3CR1GFP/GFP (orange) slices, at t0. Note that CX3CR1GFP/GFP processes are significantly more distant than CX3CR1+/GFP ones (∗p < 0.05, Kolmogorov-Smirnov test). (B) Left, plot of spatial x-y coordinates of CX3CR1+/GFP (purple symbols, n = 209/8) and CX3CR1GFP/GFP (orange symbols, n = 161/8) microglial processes 45 min after ATP application (t45). Right, cumulative distributions of radial distances of microglial processes in CX3CR1+/GFP (purple) or CX3CR1GFP/GFP (orange) slices, at t45. Note that at t45 the majority of CX3CR1GFP/GFP processes do not reach the ATP pipette (∗∗p < 0.0001, Kolmogorov-Smirnov test). (C) Box charts of displacement (left) and directionality (right) of CX3CR1+/GFP (purple) and CX3CR1GFP/GFP (orange) microglial processes. In slices from Cx3cr1+/GFP mice displacement and directionality are significantly higher than in those from Cx3cr1GFP/GFP mice (∗∗∗p < 0.0001, t-test), (D) Correlation plot showing mean instantaneous velocities vs. radial distance of CX3CR1+/GFP (purple) and CX3CR1GFP/GFP (orange) microglial processes. The velocity of CX3CR1+/GFP processes increase significantly 7 mm far from the ATP pipette tip (∗p < 0.001, Kruskal-Wallis One Way ANOVA on Ranks, and p < 0.05 multiple comparison Dunn’s method versus 15, 25, and 35 μm radial distance). Note that in the proximity of the ATP pipette, the velocity of CX3CR1GFP/GFP processes is significantly slower than that of CX3CR1+/GFP processes (#p < 0.01 t-test).

Mentions: To understand the basis of the observed difference in processes extension between the two genotypes, we performed a tracking analysis of single microglial processes (Figure 3B; movies 1 and 2 in Supplementary Material). At the start of fluorescence monitoring (t0), the number of detected processes was significantly higher in CX3CR1+/GFP than in CX3CR1GFP/GFP slices (p < 0.01, t-test; Figure 3C). Moreover, at t0 the process positions in CX3CR1GFP/GFP slices (orange symbols, Figure 4A) were more distant from the ATP-containing pipette, compared to CX3CR1+/GFP (purple, p < 0.05, Kolmogorov-Smirnov test). The observed differences may reflect the reduced ramification of CX3CR1GFP/GFP microglia, as well as their lower cell density (Paolicelli et al., 2011). Besides, in both genotypes the number of traced processes increased during the experiments, which was significantly higher 45 min after ATP puff (t45; Figure 3C). We noticed that the mean velocity of processes elongation was similar in the two genotypes (CX3CR1+/GFP 2.39 ± 0.05 μm/min, n = 210 processes/8 fields/4 mice; CX3CR1GFP/GFP 2.48 ± 0.08, n = 161/8/4; p = 0.36; not shown). Nevertheless, the distribution of microglial processes positions was remarkably different between the two genotypes also after ATP-induced extension, as CX3CR1+/GFP processes were significantly closer to the pipette tip at t45 (Figure 4B; p < 0.0001, Kolmogorov-Smirnov test). This was likely due to the lower directionality shown by microglial processes in their elongation. As shown in Figure 4C, CX3CR1GFP/GFP processes displayed lower displacement and directionality toward ATP source.


Defective microglial development in the hippocampus of Cx3cr1 deficient mice.

Pagani F, Paolicelli RC, Murana E, Cortese B, Di Angelantonio S, Zurolo E, Guiducci E, Ferreira TA, Garofalo S, Catalano M, D'Alessandro G, Porzia A, Peruzzi G, Mainiero F, Limatola C, Gross CT, Ragozzino D - Front Cell Neurosci (2015)

Tracking analysis of single processes in CX3CR1+/GFP and CX3CR1GFP/GFP microglia. (A) Left, plot of spatial x-y coordinates respect to the ATP pipette tip of microglial processes in CX3CR1+/GFP (purple symbols, n = 133 processes/8 fields) and CX3CR1GFP/GFP hippocampal slices (orange symbols, n = 89/8) at start of recordings (t0). Right, cumulative distributions of radial distances of microglial processes from the pipette tip in CX3CR1+/GFP (purple) or CX3CR1GFP/GFP (orange) slices, at t0. Note that CX3CR1GFP/GFP processes are significantly more distant than CX3CR1+/GFP ones (∗p < 0.05, Kolmogorov-Smirnov test). (B) Left, plot of spatial x-y coordinates of CX3CR1+/GFP (purple symbols, n = 209/8) and CX3CR1GFP/GFP (orange symbols, n = 161/8) microglial processes 45 min after ATP application (t45). Right, cumulative distributions of radial distances of microglial processes in CX3CR1+/GFP (purple) or CX3CR1GFP/GFP (orange) slices, at t45. Note that at t45 the majority of CX3CR1GFP/GFP processes do not reach the ATP pipette (∗∗p < 0.0001, Kolmogorov-Smirnov test). (C) Box charts of displacement (left) and directionality (right) of CX3CR1+/GFP (purple) and CX3CR1GFP/GFP (orange) microglial processes. In slices from Cx3cr1+/GFP mice displacement and directionality are significantly higher than in those from Cx3cr1GFP/GFP mice (∗∗∗p < 0.0001, t-test), (D) Correlation plot showing mean instantaneous velocities vs. radial distance of CX3CR1+/GFP (purple) and CX3CR1GFP/GFP (orange) microglial processes. The velocity of CX3CR1+/GFP processes increase significantly 7 mm far from the ATP pipette tip (∗p < 0.001, Kruskal-Wallis One Way ANOVA on Ranks, and p < 0.05 multiple comparison Dunn’s method versus 15, 25, and 35 μm radial distance). Note that in the proximity of the ATP pipette, the velocity of CX3CR1GFP/GFP processes is significantly slower than that of CX3CR1+/GFP processes (#p < 0.01 t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4379915&req=5

Figure 4: Tracking analysis of single processes in CX3CR1+/GFP and CX3CR1GFP/GFP microglia. (A) Left, plot of spatial x-y coordinates respect to the ATP pipette tip of microglial processes in CX3CR1+/GFP (purple symbols, n = 133 processes/8 fields) and CX3CR1GFP/GFP hippocampal slices (orange symbols, n = 89/8) at start of recordings (t0). Right, cumulative distributions of radial distances of microglial processes from the pipette tip in CX3CR1+/GFP (purple) or CX3CR1GFP/GFP (orange) slices, at t0. Note that CX3CR1GFP/GFP processes are significantly more distant than CX3CR1+/GFP ones (∗p < 0.05, Kolmogorov-Smirnov test). (B) Left, plot of spatial x-y coordinates of CX3CR1+/GFP (purple symbols, n = 209/8) and CX3CR1GFP/GFP (orange symbols, n = 161/8) microglial processes 45 min after ATP application (t45). Right, cumulative distributions of radial distances of microglial processes in CX3CR1+/GFP (purple) or CX3CR1GFP/GFP (orange) slices, at t45. Note that at t45 the majority of CX3CR1GFP/GFP processes do not reach the ATP pipette (∗∗p < 0.0001, Kolmogorov-Smirnov test). (C) Box charts of displacement (left) and directionality (right) of CX3CR1+/GFP (purple) and CX3CR1GFP/GFP (orange) microglial processes. In slices from Cx3cr1+/GFP mice displacement and directionality are significantly higher than in those from Cx3cr1GFP/GFP mice (∗∗∗p < 0.0001, t-test), (D) Correlation plot showing mean instantaneous velocities vs. radial distance of CX3CR1+/GFP (purple) and CX3CR1GFP/GFP (orange) microglial processes. The velocity of CX3CR1+/GFP processes increase significantly 7 mm far from the ATP pipette tip (∗p < 0.001, Kruskal-Wallis One Way ANOVA on Ranks, and p < 0.05 multiple comparison Dunn’s method versus 15, 25, and 35 μm radial distance). Note that in the proximity of the ATP pipette, the velocity of CX3CR1GFP/GFP processes is significantly slower than that of CX3CR1+/GFP processes (#p < 0.01 t-test).
Mentions: To understand the basis of the observed difference in processes extension between the two genotypes, we performed a tracking analysis of single microglial processes (Figure 3B; movies 1 and 2 in Supplementary Material). At the start of fluorescence monitoring (t0), the number of detected processes was significantly higher in CX3CR1+/GFP than in CX3CR1GFP/GFP slices (p < 0.01, t-test; Figure 3C). Moreover, at t0 the process positions in CX3CR1GFP/GFP slices (orange symbols, Figure 4A) were more distant from the ATP-containing pipette, compared to CX3CR1+/GFP (purple, p < 0.05, Kolmogorov-Smirnov test). The observed differences may reflect the reduced ramification of CX3CR1GFP/GFP microglia, as well as their lower cell density (Paolicelli et al., 2011). Besides, in both genotypes the number of traced processes increased during the experiments, which was significantly higher 45 min after ATP puff (t45; Figure 3C). We noticed that the mean velocity of processes elongation was similar in the two genotypes (CX3CR1+/GFP 2.39 ± 0.05 μm/min, n = 210 processes/8 fields/4 mice; CX3CR1GFP/GFP 2.48 ± 0.08, n = 161/8/4; p = 0.36; not shown). Nevertheless, the distribution of microglial processes positions was remarkably different between the two genotypes also after ATP-induced extension, as CX3CR1+/GFP processes were significantly closer to the pipette tip at t45 (Figure 4B; p < 0.0001, Kolmogorov-Smirnov test). This was likely due to the lower directionality shown by microglial processes in their elongation. As shown in Figure 4C, CX3CR1GFP/GFP processes displayed lower displacement and directionality toward ATP source.

Bottom Line: We found that fractalkine signaling is necessary for the development of several morphological and physiological features of microglia.Fractalkine signaling also influenced microglial morphology and ability to extend processes in response to ATP following its focal application to the slice.Our results reveal the developmental profile of several morphological and physiological properties of microglia and demonstrate that these processes are modulated by fractalkine signaling.

View Article: PubMed Central - PubMed

Affiliation: Center for Life Nanoscience - Istituto Italiano di Tecnologia@Sapienza, Rome Italy.

ABSTRACT
Microglial cells participate in brain development and influence neuronal loss and synaptic maturation. Fractalkine is an important neuronal chemokine whose expression increases during development and that can influence microglia function via the fractalkine receptor, CX3CR1. Mice lacking Cx3cr1 show a variety of neuronal defects thought to be the result of deficient microglia function. Activation of CX3CR1 is important for the proper migration of microglia to sites of injury and into the brain during development. However, little is known about how fractalkine modulates microglial properties during development. Here we examined microglial morphology, response to ATP, and K(+) current properties in acute brain slices from Cx3cr1 knockout mice across postnatal hippocampal development. We found that fractalkine signaling is necessary for the development of several morphological and physiological features of microglia. Specifically, we found that the occurrence of an outward rectifying K(+) current, typical of activated microglia, that peaked during the second and third postnatal week, was reduced in Cx3cr1 knockout mice. Fractalkine signaling also influenced microglial morphology and ability to extend processes in response to ATP following its focal application to the slice. Our results reveal the developmental profile of several morphological and physiological properties of microglia and demonstrate that these processes are modulated by fractalkine signaling.

No MeSH data available.


Related in: MedlinePlus