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In vivo characterization of microglial engulfment of dying neurons in the zebrafish spinal cord.

Morsch M, Radford R, Lee A, Don EK, Badrock AP, Hall TE, Cole NJ, Chung R - Front Cell Neurosci (2015)

Bottom Line: In vivo imaging confirmed the motile nature of microglia within the uninjured spinal cord.This process of microglial engulfment is highly dynamic, involving the extension of processes toward the lesion site and consequently the ingestion of the dying neuron. 3D rendering analysis of time-lapse recordings revealed the formation of phagosome-like structures in the activated microglia located at the site of neuronal ablation.This real-time representation of microglial phagocytosis in the living zebrafish spinal cord provides novel opportunities to study the mechanisms of microglia-mediated neuronal clearance.

View Article: PubMed Central - PubMed

Affiliation: Motor Neuron Disease Research Group, Faculty of Medicine and Health Sciences, Macquarie University Sydney, NSW, Australia.

ABSTRACT
Microglia are specialized phagocytes in the vertebrate central nervous system (CNS). As the resident immune cells of the CNS they play an important role in the removal of dying neurons during both development and in several neuronal pathologies. Microglia have been shown to prevent the diffusion of damaging degradation products of dying neurons by engulfment and ingestion. Here we describe a live imaging approach that uses UV laser ablation to selectively stress and kill spinal neurons and visualize the clearance of neuronal remnants by microglia in the zebrafish spinal cord. In vivo imaging confirmed the motile nature of microglia within the uninjured spinal cord. However, selective neuronal ablation triggered rapid activation of microglia, leading to phagocytic uptake of neuronal debris by microglia within 20-30 min. This process of microglial engulfment is highly dynamic, involving the extension of processes toward the lesion site and consequently the ingestion of the dying neuron. 3D rendering analysis of time-lapse recordings revealed the formation of phagosome-like structures in the activated microglia located at the site of neuronal ablation. This real-time representation of microglial phagocytosis in the living zebrafish spinal cord provides novel opportunities to study the mechanisms of microglia-mediated neuronal clearance.

No MeSH data available.


Related in: MedlinePlus

Microglia rapidly respond and are recruited to the site of neuronal ablation. UV ablation of a GFP-expressing spinal neuron (A; arrowhead) resulted in only one of two surrounding microglia (red) relocating and phagocytosing the dying neuron (B,C). Over a period of several hours, other microglia pass by the phagocytosing microglia (D), and after approximately 2.5 h the phagocytosing glial cell changes morphology back to a stellate morphology (E), indicating termination of the phagocytosis process (F,G). Supplementary Video 4 shows the time-lapse video of this process. Scale bars = 50 μm. (H–K) Quantitative analysis of morphometric changes in microglial response to dying spinal neurons. Microglia undergo significant changes in morphology (I–K) and speed (H) upon activation. *p < 0.05 paired Student's t-test; n = 6–7; N = 6–7; 2D ImageJ analysis (H); ***p < 0.001 unpaired Student's t-test; n = 7–22; N = 6–9; 2D ImageJ analysis (I); *p < 0.05; **p < 0.01 unpaired Student's t-test; n = 4–13; N = 4–10; 3D Imaris analysis (J,K).
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Figure 6: Microglia rapidly respond and are recruited to the site of neuronal ablation. UV ablation of a GFP-expressing spinal neuron (A; arrowhead) resulted in only one of two surrounding microglia (red) relocating and phagocytosing the dying neuron (B,C). Over a period of several hours, other microglia pass by the phagocytosing microglia (D), and after approximately 2.5 h the phagocytosing glial cell changes morphology back to a stellate morphology (E), indicating termination of the phagocytosis process (F,G). Supplementary Video 4 shows the time-lapse video of this process. Scale bars = 50 μm. (H–K) Quantitative analysis of morphometric changes in microglial response to dying spinal neurons. Microglia undergo significant changes in morphology (I–K) and speed (H) upon activation. *p < 0.05 paired Student's t-test; n = 6–7; N = 6–7; 2D ImageJ analysis (H); ***p < 0.001 unpaired Student's t-test; n = 7–22; N = 6–9; 2D ImageJ analysis (I); *p < 0.05; **p < 0.01 unpaired Student's t-test; n = 4–13; N = 4–10; 3D Imaris analysis (J,K).

Mentions: In order to characterize the phagocytosis of dying neurons via microglial engulfment in the spinal cord, we generated transgenic lines in which zebrafish expressed together green fluorescent neurons (islet1:GFP) and a subset of red microglia (mpeg1:mCherry; Figures 1, 5, 6). The microglial population in these fish showed a ramified morphology that was reminiscent of reports from the (fish) brain (Peri and Nüsslein-Volhard, 2008; Svahn et al., 2013). Consistent with our mpeg1 fish line that expressed fluorescence in a subset of microglia (Gal4:UAS), UV laser ablation of some neurons did not lead to a response of the fluorescent microglia. In approximately 60% of our ablations we did not observe subsequent microglia engulfment, conceivably because non-fluorescent microglia would have responded to ensure the immediate uptake of the neuronal debris. In successful experiments, time-lapse imaging revealed that soon after UV laser ablation of a single motor neuron, individual microglia underwent dramatic changes in morphology by extending and retracting processes, moving toward the site of the ablated neuron and changing to a spherical shape within minutes (Figure 6). The typical first response was for microglia to extend phagocytic protrusions toward the ablated neuron body, therefore shifting the whole microglia body toward the lesion site. This process took on average 27 min until complete engulfment of the ablated soma was achieved (Table 1). Notably, this process was characterized by a remarkable increase in the dynamic behavior of the microglia as it moved toward the dying neuron (Supplementary Video 4). Within minutes, activated microglia doubled their speed (2.7 μm/min vs. 1.5 μm/min in the “surveying” state; Table 1; Figures 6H–K), covered substantial distances of 33–94 μm toward the lesion site (72.8 μm on average), and decreased in size significantly to form a round amoeboid body as it engulfed the neuron remnants (128.9 μm2 vs. 229.4 μm2 before activation; Table 1; Figures 6H–K).


In vivo characterization of microglial engulfment of dying neurons in the zebrafish spinal cord.

Morsch M, Radford R, Lee A, Don EK, Badrock AP, Hall TE, Cole NJ, Chung R - Front Cell Neurosci (2015)

Microglia rapidly respond and are recruited to the site of neuronal ablation. UV ablation of a GFP-expressing spinal neuron (A; arrowhead) resulted in only one of two surrounding microglia (red) relocating and phagocytosing the dying neuron (B,C). Over a period of several hours, other microglia pass by the phagocytosing microglia (D), and after approximately 2.5 h the phagocytosing glial cell changes morphology back to a stellate morphology (E), indicating termination of the phagocytosis process (F,G). Supplementary Video 4 shows the time-lapse video of this process. Scale bars = 50 μm. (H–K) Quantitative analysis of morphometric changes in microglial response to dying spinal neurons. Microglia undergo significant changes in morphology (I–K) and speed (H) upon activation. *p < 0.05 paired Student's t-test; n = 6–7; N = 6–7; 2D ImageJ analysis (H); ***p < 0.001 unpaired Student's t-test; n = 7–22; N = 6–9; 2D ImageJ analysis (I); *p < 0.05; **p < 0.01 unpaired Student's t-test; n = 4–13; N = 4–10; 3D Imaris analysis (J,K).
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Figure 6: Microglia rapidly respond and are recruited to the site of neuronal ablation. UV ablation of a GFP-expressing spinal neuron (A; arrowhead) resulted in only one of two surrounding microglia (red) relocating and phagocytosing the dying neuron (B,C). Over a period of several hours, other microglia pass by the phagocytosing microglia (D), and after approximately 2.5 h the phagocytosing glial cell changes morphology back to a stellate morphology (E), indicating termination of the phagocytosis process (F,G). Supplementary Video 4 shows the time-lapse video of this process. Scale bars = 50 μm. (H–K) Quantitative analysis of morphometric changes in microglial response to dying spinal neurons. Microglia undergo significant changes in morphology (I–K) and speed (H) upon activation. *p < 0.05 paired Student's t-test; n = 6–7; N = 6–7; 2D ImageJ analysis (H); ***p < 0.001 unpaired Student's t-test; n = 7–22; N = 6–9; 2D ImageJ analysis (I); *p < 0.05; **p < 0.01 unpaired Student's t-test; n = 4–13; N = 4–10; 3D Imaris analysis (J,K).
Mentions: In order to characterize the phagocytosis of dying neurons via microglial engulfment in the spinal cord, we generated transgenic lines in which zebrafish expressed together green fluorescent neurons (islet1:GFP) and a subset of red microglia (mpeg1:mCherry; Figures 1, 5, 6). The microglial population in these fish showed a ramified morphology that was reminiscent of reports from the (fish) brain (Peri and Nüsslein-Volhard, 2008; Svahn et al., 2013). Consistent with our mpeg1 fish line that expressed fluorescence in a subset of microglia (Gal4:UAS), UV laser ablation of some neurons did not lead to a response of the fluorescent microglia. In approximately 60% of our ablations we did not observe subsequent microglia engulfment, conceivably because non-fluorescent microglia would have responded to ensure the immediate uptake of the neuronal debris. In successful experiments, time-lapse imaging revealed that soon after UV laser ablation of a single motor neuron, individual microglia underwent dramatic changes in morphology by extending and retracting processes, moving toward the site of the ablated neuron and changing to a spherical shape within minutes (Figure 6). The typical first response was for microglia to extend phagocytic protrusions toward the ablated neuron body, therefore shifting the whole microglia body toward the lesion site. This process took on average 27 min until complete engulfment of the ablated soma was achieved (Table 1). Notably, this process was characterized by a remarkable increase in the dynamic behavior of the microglia as it moved toward the dying neuron (Supplementary Video 4). Within minutes, activated microglia doubled their speed (2.7 μm/min vs. 1.5 μm/min in the “surveying” state; Table 1; Figures 6H–K), covered substantial distances of 33–94 μm toward the lesion site (72.8 μm on average), and decreased in size significantly to form a round amoeboid body as it engulfed the neuron remnants (128.9 μm2 vs. 229.4 μm2 before activation; Table 1; Figures 6H–K).

Bottom Line: In vivo imaging confirmed the motile nature of microglia within the uninjured spinal cord.This process of microglial engulfment is highly dynamic, involving the extension of processes toward the lesion site and consequently the ingestion of the dying neuron. 3D rendering analysis of time-lapse recordings revealed the formation of phagosome-like structures in the activated microglia located at the site of neuronal ablation.This real-time representation of microglial phagocytosis in the living zebrafish spinal cord provides novel opportunities to study the mechanisms of microglia-mediated neuronal clearance.

View Article: PubMed Central - PubMed

Affiliation: Motor Neuron Disease Research Group, Faculty of Medicine and Health Sciences, Macquarie University Sydney, NSW, Australia.

ABSTRACT
Microglia are specialized phagocytes in the vertebrate central nervous system (CNS). As the resident immune cells of the CNS they play an important role in the removal of dying neurons during both development and in several neuronal pathologies. Microglia have been shown to prevent the diffusion of damaging degradation products of dying neurons by engulfment and ingestion. Here we describe a live imaging approach that uses UV laser ablation to selectively stress and kill spinal neurons and visualize the clearance of neuronal remnants by microglia in the zebrafish spinal cord. In vivo imaging confirmed the motile nature of microglia within the uninjured spinal cord. However, selective neuronal ablation triggered rapid activation of microglia, leading to phagocytic uptake of neuronal debris by microglia within 20-30 min. This process of microglial engulfment is highly dynamic, involving the extension of processes toward the lesion site and consequently the ingestion of the dying neuron. 3D rendering analysis of time-lapse recordings revealed the formation of phagosome-like structures in the activated microglia located at the site of neuronal ablation. This real-time representation of microglial phagocytosis in the living zebrafish spinal cord provides novel opportunities to study the mechanisms of microglia-mediated neuronal clearance.

No MeSH data available.


Related in: MedlinePlus