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Astrocytes contribute to synapse elimination via type 2 inositol 1,4,5-trisphosphate receptor-dependent release of ATP.

Yang J, Yang H, Liu Y, Li X, Qin L, Lou H, Duan S, Wang H - Elife (2016)

Bottom Line: Selective elimination of unwanted synapses is vital for the precise formation of neuronal circuits during development, but the underlying mechanisms remain unclear.Interestingly, intracerebroventricular injection of ATP, but not adenosine, rescued the deficit in synapse elimination in Itpr2(-/-) mice.Our results uncovered a novel mechanism suggesting that astrocytes release ATP in an IP3R2-dependent manner to regulate synapse elimination.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Key Laboratory of Medical Neurobiology of Ministry of Health of China, Key Laboratory of Neurobiology, Zhejiang University School of Medicine, Hangzhou, China.

ABSTRACT
Selective elimination of unwanted synapses is vital for the precise formation of neuronal circuits during development, but the underlying mechanisms remain unclear. Using inositol 1,4,5-trisphosphate receptor type 2 knockout (Itpr2(-/-)) mice to specifically disturb somatic Ca(2+) signaling in astrocytes, we showed that developmental elimination of the ventral posteromedial nucleus relay synapse was impaired. Interestingly, intracerebroventricular injection of ATP, but not adenosine, rescued the deficit in synapse elimination in Itpr2(-/-) mice. Further studies showed that developmental synapse elimination was also impaired in P2ry1(-/-) mice and was not rescued by ATP, indicating a possible role of purinergic signaling. This hypothesis was confirmed by MRS-2365, a selective P2Y1 agonist, could also rescue the deficient of synapse elimination in Itpr2(-/-) mice. Our results uncovered a novel mechanism suggesting that astrocytes release ATP in an IP3R2-dependent manner to regulate synapse elimination.

No MeSH data available.


Related in: MedlinePlus

Injury-induced inflammatory responses of astrocytes and microglia are equivalent in aCSF and ATP treated-Itpr2−/− mice.(a) Confocal image of a coronal brain section stained with DAPI showing the location of cannula. White dotted line indicates the cannula. Scale bar, 100 µM. (b) Representative confocal images illustrating reactive astrocyte and activated microglia around the site of cannula placement in aCSF and ATP treated-Itpr2−/− mice. Astrocytes were labeled by the specific marker GFAP (green). Microglias were visualized by Iba1 staining (red). Scale bar, 20 µM. (c-e) Histogram summary of the max reactive distance from the cannula (c, n = 6 mice per group, p=0.93), the number of reactive astrocytes (d, n = 8 sections from 4 mice per group, p=0.96), and the percentage of activated microglia (e, n = 7 sections from 4 mice per group, p=0.09) near the site of cannula in aCSF and ATP treated-Itpr2−/− mice. Error bars indicate SEM.DOI:http://dx.doi.org/10.7554/eLife.15043.011
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fig3s1: Injury-induced inflammatory responses of astrocytes and microglia are equivalent in aCSF and ATP treated-Itpr2−/− mice.(a) Confocal image of a coronal brain section stained with DAPI showing the location of cannula. White dotted line indicates the cannula. Scale bar, 100 µM. (b) Representative confocal images illustrating reactive astrocyte and activated microglia around the site of cannula placement in aCSF and ATP treated-Itpr2−/− mice. Astrocytes were labeled by the specific marker GFAP (green). Microglias were visualized by Iba1 staining (red). Scale bar, 20 µM. (c-e) Histogram summary of the max reactive distance from the cannula (c, n = 6 mice per group, p=0.93), the number of reactive astrocytes (d, n = 8 sections from 4 mice per group, p=0.96), and the percentage of activated microglia (e, n = 7 sections from 4 mice per group, p=0.09) near the site of cannula in aCSF and ATP treated-Itpr2−/− mice. Error bars indicate SEM.DOI:http://dx.doi.org/10.7554/eLife.15043.011

Mentions: Increasing evidence suggests that astrocytes release a number of gliotransmitters, such as glutamate, ATP, and D-serine to regulate synaptic transmission and synaptic plasticity (Chen et al., 2013; Henneberger et al., 2010; Jourdain et al., 2007). Among these gliotransmitters, we were particularly interested in ATP and assumed that it might play a role in synapse elimination for the following reasons: 1) the basal ATP levels are reduced in Itpr2−/− mice (Cao et al., 2013); 2) astrocytes release ATP in a Ca2+-dependent manner (Lalo et al., 2014; Zhang et al., 2007); and 3) ATP regulates synaptic plasticity (Chen et al., 2013). We then tested whether the basal ATP level in the VPm changes during development at the time when exploratory activity increases in WT mice. A significant increase in the basal ATP level occurred at P18 compared with that at P7 in WT mice (Figure 3a). This developmental up-regulation of the basal ATP level was absent in Itpr2−/− mice (Figure 3a). In addition, we found that the basal ATP level was comparable between WT and Itpr2−/− mice at P7 but was significantly reduced in KO mice at P18 (Figure 3a). These results suggested that, during development, the basal ATP level increases in WT but not in Itpr2−/− mice. Next, we tested whether the deficit of synapse elimination in Itpr2−/− mice could be rescued by compensatory ATP. To achieve this goal, we implanted a cannula in the left lateral ventricular of the brain (Figure 3b) and found that intracerebroventricular injection of ATP from P11 to P15 rescued the synapse elimination deficit (Figure 3e,i,l,m). At P16-17, unlike aCSF-treated mice, the majority of VPm neurons in Itpr2−/− mice that had received ATP (50 µM) treatment was innervated by a single Pr5 input (Figure 3l). Immunostaining for VGluT2 also revealed that the number of synapses significantly decreased in Itpr2−/− mice with ATP treatment (Figure 3p,q). These results indicate that ATP treatment is sufficient to rescue synapse elimination deficit in Itpr2−/− mice and the removal of redundant synapses is independent of IP3R2-dependent Ca2+ signaling. Cannula implantation caused injury may induce inflammatory responses of astrocytes and microglia differentially between aCSF and ATP treated-Itpr2−/− mice, thereby affected the rescue effects. We found that reactive astrocytes and activated microglia around the site of cannula placement in aCSF and ATP treated-Itpr2−/−mice were identical (Figure 3—figure supplement 1), and ruled out this possibility. We next applied a low dose ATP (5 µM) and found that the impaired synapse elimination cannot be rescued in Itpr2−/− mice, suggesting the rescue effect of ATP was in a dose-dependent manner (Figure 3n,o).10.7554/eLife.15043.010Figure 3.Intracerebroventricular injection of ATP from P11 to P15 rescued the synapse elimination deficit in Itpr2−/− mice.


Astrocytes contribute to synapse elimination via type 2 inositol 1,4,5-trisphosphate receptor-dependent release of ATP.

Yang J, Yang H, Liu Y, Li X, Qin L, Lou H, Duan S, Wang H - Elife (2016)

Injury-induced inflammatory responses of astrocytes and microglia are equivalent in aCSF and ATP treated-Itpr2−/− mice.(a) Confocal image of a coronal brain section stained with DAPI showing the location of cannula. White dotted line indicates the cannula. Scale bar, 100 µM. (b) Representative confocal images illustrating reactive astrocyte and activated microglia around the site of cannula placement in aCSF and ATP treated-Itpr2−/− mice. Astrocytes were labeled by the specific marker GFAP (green). Microglias were visualized by Iba1 staining (red). Scale bar, 20 µM. (c-e) Histogram summary of the max reactive distance from the cannula (c, n = 6 mice per group, p=0.93), the number of reactive astrocytes (d, n = 8 sections from 4 mice per group, p=0.96), and the percentage of activated microglia (e, n = 7 sections from 4 mice per group, p=0.09) near the site of cannula in aCSF and ATP treated-Itpr2−/− mice. Error bars indicate SEM.DOI:http://dx.doi.org/10.7554/eLife.15043.011
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fig3s1: Injury-induced inflammatory responses of astrocytes and microglia are equivalent in aCSF and ATP treated-Itpr2−/− mice.(a) Confocal image of a coronal brain section stained with DAPI showing the location of cannula. White dotted line indicates the cannula. Scale bar, 100 µM. (b) Representative confocal images illustrating reactive astrocyte and activated microglia around the site of cannula placement in aCSF and ATP treated-Itpr2−/− mice. Astrocytes were labeled by the specific marker GFAP (green). Microglias were visualized by Iba1 staining (red). Scale bar, 20 µM. (c-e) Histogram summary of the max reactive distance from the cannula (c, n = 6 mice per group, p=0.93), the number of reactive astrocytes (d, n = 8 sections from 4 mice per group, p=0.96), and the percentage of activated microglia (e, n = 7 sections from 4 mice per group, p=0.09) near the site of cannula in aCSF and ATP treated-Itpr2−/− mice. Error bars indicate SEM.DOI:http://dx.doi.org/10.7554/eLife.15043.011
Mentions: Increasing evidence suggests that astrocytes release a number of gliotransmitters, such as glutamate, ATP, and D-serine to regulate synaptic transmission and synaptic plasticity (Chen et al., 2013; Henneberger et al., 2010; Jourdain et al., 2007). Among these gliotransmitters, we were particularly interested in ATP and assumed that it might play a role in synapse elimination for the following reasons: 1) the basal ATP levels are reduced in Itpr2−/− mice (Cao et al., 2013); 2) astrocytes release ATP in a Ca2+-dependent manner (Lalo et al., 2014; Zhang et al., 2007); and 3) ATP regulates synaptic plasticity (Chen et al., 2013). We then tested whether the basal ATP level in the VPm changes during development at the time when exploratory activity increases in WT mice. A significant increase in the basal ATP level occurred at P18 compared with that at P7 in WT mice (Figure 3a). This developmental up-regulation of the basal ATP level was absent in Itpr2−/− mice (Figure 3a). In addition, we found that the basal ATP level was comparable between WT and Itpr2−/− mice at P7 but was significantly reduced in KO mice at P18 (Figure 3a). These results suggested that, during development, the basal ATP level increases in WT but not in Itpr2−/− mice. Next, we tested whether the deficit of synapse elimination in Itpr2−/− mice could be rescued by compensatory ATP. To achieve this goal, we implanted a cannula in the left lateral ventricular of the brain (Figure 3b) and found that intracerebroventricular injection of ATP from P11 to P15 rescued the synapse elimination deficit (Figure 3e,i,l,m). At P16-17, unlike aCSF-treated mice, the majority of VPm neurons in Itpr2−/− mice that had received ATP (50 µM) treatment was innervated by a single Pr5 input (Figure 3l). Immunostaining for VGluT2 also revealed that the number of synapses significantly decreased in Itpr2−/− mice with ATP treatment (Figure 3p,q). These results indicate that ATP treatment is sufficient to rescue synapse elimination deficit in Itpr2−/− mice and the removal of redundant synapses is independent of IP3R2-dependent Ca2+ signaling. Cannula implantation caused injury may induce inflammatory responses of astrocytes and microglia differentially between aCSF and ATP treated-Itpr2−/− mice, thereby affected the rescue effects. We found that reactive astrocytes and activated microglia around the site of cannula placement in aCSF and ATP treated-Itpr2−/−mice were identical (Figure 3—figure supplement 1), and ruled out this possibility. We next applied a low dose ATP (5 µM) and found that the impaired synapse elimination cannot be rescued in Itpr2−/− mice, suggesting the rescue effect of ATP was in a dose-dependent manner (Figure 3n,o).10.7554/eLife.15043.010Figure 3.Intracerebroventricular injection of ATP from P11 to P15 rescued the synapse elimination deficit in Itpr2−/− mice.

Bottom Line: Selective elimination of unwanted synapses is vital for the precise formation of neuronal circuits during development, but the underlying mechanisms remain unclear.Interestingly, intracerebroventricular injection of ATP, but not adenosine, rescued the deficit in synapse elimination in Itpr2(-/-) mice.Our results uncovered a novel mechanism suggesting that astrocytes release ATP in an IP3R2-dependent manner to regulate synapse elimination.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Key Laboratory of Medical Neurobiology of Ministry of Health of China, Key Laboratory of Neurobiology, Zhejiang University School of Medicine, Hangzhou, China.

ABSTRACT
Selective elimination of unwanted synapses is vital for the precise formation of neuronal circuits during development, but the underlying mechanisms remain unclear. Using inositol 1,4,5-trisphosphate receptor type 2 knockout (Itpr2(-/-)) mice to specifically disturb somatic Ca(2+) signaling in astrocytes, we showed that developmental elimination of the ventral posteromedial nucleus relay synapse was impaired. Interestingly, intracerebroventricular injection of ATP, but not adenosine, rescued the deficit in synapse elimination in Itpr2(-/-) mice. Further studies showed that developmental synapse elimination was also impaired in P2ry1(-/-) mice and was not rescued by ATP, indicating a possible role of purinergic signaling. This hypothesis was confirmed by MRS-2365, a selective P2Y1 agonist, could also rescue the deficient of synapse elimination in Itpr2(-/-) mice. Our results uncovered a novel mechanism suggesting that astrocytes release ATP in an IP3R2-dependent manner to regulate synapse elimination.

No MeSH data available.


Related in: MedlinePlus