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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

Confocal images showing that IP3R2 was specifically expressed in GFAP-positive astrocytes but not GFP-positive microglia.Microglias were visualized using transgenic mice in which all microglia express GFP (green) under the control of the CX3CR1 promoter. Astrocytes were labeled by the specific marker GFAP (blue). IP3R2 was co-localized with GFAP-positive astrocytes (arrowheads) but not GFP- positive microglia (arrows). Scale bar, 20 µm. Images were obtained from hippocampus but not the VPm because the same antibodies (Santa cruz, sc-7278; Millipore, AB3000) that worked for hippocampus did not work for VPm.DOI:http://dx.doi.org/10.7554/eLife.15043.004
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fig1s1: Confocal images showing that IP3R2 was specifically expressed in GFAP-positive astrocytes but not GFP-positive microglia.Microglias were visualized using transgenic mice in which all microglia express GFP (green) under the control of the CX3CR1 promoter. Astrocytes were labeled by the specific marker GFAP (blue). IP3R2 was co-localized with GFAP-positive astrocytes (arrowheads) but not GFP- positive microglia (arrows). Scale bar, 20 µm. Images were obtained from hippocampus but not the VPm because the same antibodies (Santa cruz, sc-7278; Millipore, AB3000) that worked for hippocampus did not work for VPm.DOI:http://dx.doi.org/10.7554/eLife.15043.004

Mentions: To determine the role of astrocytic Ca2+ signaling in synapse elimination, we need an effective way to selectively disrupt the function of astrocytes. In mammals, astrocytes rely on IP3R2-mediated intracellular Ca2+ signaling to perform their functions. A large literature suggested that IP3R2 is the only subtype astrocytes expressed and is not expressed in microglia and neurons in the cerebrum. Thus Itpr2−/− mice could be used to study the specific roles of Ca2+ signaling in astrocytes for synapse function (Hertle and Yeckel, 2007; Li et al., 2015; Sharp et al., 1999). In line with previous studies, we also found that IP3R2 was co-expressed with GFAP, but not bio-markers for microglia or neurons in the brain areas we tested including hippocampus (Figure 1—figure supplement 1). Using Ca2+ imaging in acute brain slices, we next found that the ATP -induced somatic [Ca2+]i elevation in the astrocytes but not in the neurons of Itpr2−/− mice (Figure 1—figure supplement 2b,c) was abolished in both of the VPm and hippocampus, confirming that Ca2+ signaling was selectively impaired in astrocytes in Itpr2−/− mice. Next, we examined developmental synapse elimination by whole-cell patch recording in acute brain slices. Interestingly, we found a marked difference in the mean number of inputs received by each VPm neuron between WT and Itpr2−/− mice at P16-18 (WT = 1.2 ± 0.02, n = 26 cells from 4 mice; Itpr2−/− = 2.1 ± 0.10, n = 40 cells from 6 mice; p<0.01, Figure 1d). In WT mice, only 27% (7 of 26) of VPm relay neurons received multiple Pr5 inputs at this age (Figure 1a,c), whereas most of these neurons (72%, 32 of 42) in Itpr2−/− mice received multiple Pr5 inputs (Figure 1b,c). VPm relay neurons receive two major excitatory inputs: from layer VI cortex and the other from the Pr5 that express vesicular glutamate transporter 1 (VGluT1) and VGluT2, respectively (Graziano et al., 2008). Each Pr5 input forms multiple synaptic contacts with VPm neurons and thus, the number of inputs indicates how many axonal projections while the VGluT2 staining represents number of synaptic terminals. The pruning of somatic innervations by Pr5 inputs in the VPm is always related to the elimination of VPm relay synapses, as showed by previous studies (Takeuchi et al., 2014; Zhang et al., 2012). To further verify that there were more synapses in KO mice, we immunostained for VGluT2. Consistent with the electrophysiological results, we observed more VGluT2 puncta around the soma as well as the total numbers of puncta in Itpr2−/− mice (Figure 1e,f), indicating that there were more synapses in KO mice. In addition, we found that neuron number did not significantly change in the VPm between WT and Itpr2−/− mice at this age (Figure 1—figure supplement 3). These results strongly suggested that astrocytic IP3R2-dependent Ca2+ signaling is required for synapse elimination.10.7554/eLife.15043.003Figure 1.Developmental synapse elimination was impaired in Itpr2−/− mice at P16-17.


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)

Confocal images showing that IP3R2 was specifically expressed in GFAP-positive astrocytes but not GFP-positive microglia.Microglias were visualized using transgenic mice in which all microglia express GFP (green) under the control of the CX3CR1 promoter. Astrocytes were labeled by the specific marker GFAP (blue). IP3R2 was co-localized with GFAP-positive astrocytes (arrowheads) but not GFP- positive microglia (arrows). Scale bar, 20 µm. Images were obtained from hippocampus but not the VPm because the same antibodies (Santa cruz, sc-7278; Millipore, AB3000) that worked for hippocampus did not work for VPm.DOI:http://dx.doi.org/10.7554/eLife.15043.004
© Copyright Policy
Related In: Results  -  Collection

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fig1s1: Confocal images showing that IP3R2 was specifically expressed in GFAP-positive astrocytes but not GFP-positive microglia.Microglias were visualized using transgenic mice in which all microglia express GFP (green) under the control of the CX3CR1 promoter. Astrocytes were labeled by the specific marker GFAP (blue). IP3R2 was co-localized with GFAP-positive astrocytes (arrowheads) but not GFP- positive microglia (arrows). Scale bar, 20 µm. Images were obtained from hippocampus but not the VPm because the same antibodies (Santa cruz, sc-7278; Millipore, AB3000) that worked for hippocampus did not work for VPm.DOI:http://dx.doi.org/10.7554/eLife.15043.004
Mentions: To determine the role of astrocytic Ca2+ signaling in synapse elimination, we need an effective way to selectively disrupt the function of astrocytes. In mammals, astrocytes rely on IP3R2-mediated intracellular Ca2+ signaling to perform their functions. A large literature suggested that IP3R2 is the only subtype astrocytes expressed and is not expressed in microglia and neurons in the cerebrum. Thus Itpr2−/− mice could be used to study the specific roles of Ca2+ signaling in astrocytes for synapse function (Hertle and Yeckel, 2007; Li et al., 2015; Sharp et al., 1999). In line with previous studies, we also found that IP3R2 was co-expressed with GFAP, but not bio-markers for microglia or neurons in the brain areas we tested including hippocampus (Figure 1—figure supplement 1). Using Ca2+ imaging in acute brain slices, we next found that the ATP -induced somatic [Ca2+]i elevation in the astrocytes but not in the neurons of Itpr2−/− mice (Figure 1—figure supplement 2b,c) was abolished in both of the VPm and hippocampus, confirming that Ca2+ signaling was selectively impaired in astrocytes in Itpr2−/− mice. Next, we examined developmental synapse elimination by whole-cell patch recording in acute brain slices. Interestingly, we found a marked difference in the mean number of inputs received by each VPm neuron between WT and Itpr2−/− mice at P16-18 (WT = 1.2 ± 0.02, n = 26 cells from 4 mice; Itpr2−/− = 2.1 ± 0.10, n = 40 cells from 6 mice; p<0.01, Figure 1d). In WT mice, only 27% (7 of 26) of VPm relay neurons received multiple Pr5 inputs at this age (Figure 1a,c), whereas most of these neurons (72%, 32 of 42) in Itpr2−/− mice received multiple Pr5 inputs (Figure 1b,c). VPm relay neurons receive two major excitatory inputs: from layer VI cortex and the other from the Pr5 that express vesicular glutamate transporter 1 (VGluT1) and VGluT2, respectively (Graziano et al., 2008). Each Pr5 input forms multiple synaptic contacts with VPm neurons and thus, the number of inputs indicates how many axonal projections while the VGluT2 staining represents number of synaptic terminals. The pruning of somatic innervations by Pr5 inputs in the VPm is always related to the elimination of VPm relay synapses, as showed by previous studies (Takeuchi et al., 2014; Zhang et al., 2012). To further verify that there were more synapses in KO mice, we immunostained for VGluT2. Consistent with the electrophysiological results, we observed more VGluT2 puncta around the soma as well as the total numbers of puncta in Itpr2−/− mice (Figure 1e,f), indicating that there were more synapses in KO mice. In addition, we found that neuron number did not significantly change in the VPm between WT and Itpr2−/− mice at this age (Figure 1—figure supplement 3). These results strongly suggested that astrocytic IP3R2-dependent Ca2+ signaling is required for synapse elimination.10.7554/eLife.15043.003Figure 1.Developmental synapse elimination was impaired in Itpr2−/− mice at P16-17.

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