Dissecting surveying behavior of reactive microglia under chronic neurodegeneration
- Department of Neurobiology, University of Maryland School of Medicine, United States
Figures
Figure 1 with 1 supplement
Iba1+ cells envelop neurons in SSLOW-infected brains.
(a) Immunostaining for microglia (IBA1, red) and neurons (NeuN, green) showing neuronal envelopment by myeloid cells in cerebral cortex of Cx3cr1/EGFP mice infected by SSLOW via intraperitoneal route at the terminal stage of the disease. (b) Immunostaining for microglia (IBA1, red) and neurons (NeuN, green) of non-infected, age-matched Cx3cr1/EGFP mice. Confocal microscopy imaging followed by 3D reconstruction was used for both a and b.
Figure 1—figure supplement 1
Prion pathogenesis is not changed in Cx3cr1/EGFP mice.
Survival curves for Cx3cr1/EGFP and WT (C57Bl/6J) mice inoculated with SSLOW via ic route (a), SSLOW via ip route (b), or 22L via ip route (c). Comparison by Mantel–Cox test. Representative Western blot image (d) and quantification of PrPSc (e) in brains of WT and Cx3cr1/EGFP mice infected via ip route. The data presented as means ± SD; p by Brown–Forsythe and Welch ANOVA with Dunnett’s multiple comparison test, n = 3 per group. Data for non-infected WT and Cx3cr1/EGFP brains (normal) are shown as a reference. Immunostaining for microglia (Iba1, green) and neurons (NeuN, red) showing reactive Iba1+ cells enveloping neurons in cortex of Cx3cr1/EGFP mice infected by SSLOW via ip route at the terminal stage of the disease (f); and lack of neuronal envelopment in adult, non-infected Cx3cr1/EGFP mice (g).
-
Figure 1—figure supplement 1—source data 1
Western blot image of brains of WT and Cx3cr1/EGFP mice infected with SSLOW and age-matched controls.
- https://cdn.elifesciences.org/articles/107650/elife-107650-fig1-figsupp1-data1-v1.zip
-
Figure 1—figure supplement 1—source data 2
Uncropped Western blot image of brains of WT and Cx3cr1/EGFP mice infected with SSLOW and age-matched controls.
- https://cdn.elifesciences.org/articles/107650/elife-107650-fig1-figsupp1-data2-v1.zip
Figure 2 with 4 supplements
Reactive myeloid cells are highly mobile.
Acute cerebral cortical slices were prepared using non-infected Cx3cr1/EGFP (normal) mice, or Cx3cr1/EGFP mice infected with SSLOW via ip route and examined at sub-clinical, early clinical, or advanced stages of the disease. Analysis of mean speed (a) and total distance (b) traveled by individual EGFP+ cells over a 3-hr period. The midline of the box-and-whisker plot denotes the median, the + represents the mean, and the ends of the box plot denote the 25th and 75th percentiles. (c) Examples of tracks recorded from EGFP+ cells. Colored lines represent tracks of individual cells recorded within a 3-hr period. (d) Principal component analysis of mobility parameters. N = 3 animals per group; n = 70–90 cells per group, *p < 0.05, **p < 0.01, ****p < 0.0001, ns – non-significant by non-parametric Kruskal–Wallis test with Dunn’s multiple comparison.
Figure 2—figure supplement 1
Accumulation of PrPSc in SSLOW-infected Cx3cr1/EGFP mice.
(a) Western blot analysis of PrPSc in brain homogenates from Cx3cr1/EGFP mice used for time-lapse imaging experiments, compared to non-infected controls. Mice were infected with SSLOW via intraperitoneal injection and analyzed at sub-clinical (111–113 dpi), early clinical (125–128 dpi), and advanced (162–169 dpi) disease stages. Non-infected mice were analyzed at 160–164 days of age. Samples were digested with Proteinase K and probed with the 3D17 anti-PrP antibody. (b) Densitometric quantification of PrPSc signal intensity. Data are shown as mean ± SD. Statistical significance was determined by ordinary one-way ANOVA with Dunnett’s multiple comparison test (*p < 0.05, ***p < 0.001, ****p < 0.0001). N = 3 mice per group.
-
Figure 2—figure supplement 1—source data 1
Western blot image of brain homogenates from prion-infected Cx3cr1/EGFP mice used for time-lapse imaging experiments and to non-infected controls.
- https://cdn.elifesciences.org/articles/107650/elife-107650-fig2-figsupp1-data1-v1.zip
-
Figure 2—figure supplement 1—source data 2
Uncropped Western blot image of brain homogenates from prion-infected Cx3cr1/EGFP mice used for time-lapse imaging experiments and to non-infected controls.
- https://cdn.elifesciences.org/articles/107650/elife-107650-fig2-figsupp1-data2-v1.zip
Figure 2—figure supplement 2
Mobility of EGFP+ cells across six consecutive 1-hr intervals post-slicing.
Acute cerebral cortical slices were prepared using non-infected Cx3cr1/EGFP (normal) mice or Cx3cr1/EGFP mice infected with SSLOW via ip route at sub-clinical and early clinical stages of the disease. Distance covered by individual EGFP+ cells in 1-hr periods across six consecutive time intervals. Means are marked by black lines. N = 3 animals per group; n = 40–65 cells per group, **p < 0.01, ****p < 0.0001, ns – non-significant by non-parametric Kruskal–Wallis test with Dunn’s multiple comparison test.
Figure 2—video 1
Time-lapse video of acute brain slices from a normal, non-infected Cx3cr1/EGFP animal.
Recording is performed over 6 hr at a rate of one frame every 5 min. Neuronal nuclei were visualized using Hoechst staining.
Figure 2—video 2
Time-lapse imaging of acute brain slices from a SSLOW-infected Cx3cr1/EGFP mouse at the sub-clinical stage of the disease.
Neuronal nuclei were visualized using Hoechst staining. Images were acquired over a 3-hr period at a rate of one frame every 5 min.
Figure 3 with 1 supplement
High- and low-mobility behavioral patterns of myeloid cells in prion-infected brains.
Acute cerebral cortical slices were prepared using normal, non-infected Cx3cr1/EGFP mice, or SSLOW-infected Cx3cr1/EGFP mice and examined at the sub-clinical, early clinical, or advanced stages of the disease. (a) Rose plot of individual cell trajectories. In brain slices from SSLOW-infected animals, EGFP+ cells showed two behavioral patterns – with high and low mobility. (b) Change in the percentage of high-mobility EGFP+ cells with the disease progression. N = 3 animals per group. The data presented as means ± SD, **p < 0.01, ****p < 0.0001, ns – non-significant by Tukey’s multiple comparisons test. Analysis of mean speed (c), total distance traveled over 3-hr period (d), and mean directional change rate (e) for individual high- and low-mobility EGFP+ cells in slices from SSLOW-infected mice at three disease stages, and normal mice. The midline of the box-and-whisker plot denotes the median, the + represents the mean, and the ends of the box plot denote the 25th and 75th percentiles. N = 3 animals per group; n = 25–30 cells per group, *p < 0.05, **p < 0.01, ***p < 0.01, ****p < 0.0001, ns – non-significant by non-parametric Kruskal–Wallis test with Dunn’s multiple comparison.
Figure 3—figure supplement 1
Inter-animal variation in high- and low-mobility behavioral phenotypes.
Mean speed (a), total distance traveled over a 3-hr period (b), and mean directional change rate (c) were quantified for high- and low-mobility EGFP+ cells in cerebral cortical slices from SSLOW-infected mice at three disease stages, and from non-infected control mice. In Superplots, colors indicate individual animals, dots represent single cells, circles show average value for each animal, black lines indicate group means calculated from biological replicates (animals). N = 3 animals per group. Significance was determined by ordinary one-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns – non-significant.
Figure 4 with 7 supplements
Behavioral patterns of high-mobility reactive myeloid cells.
Time-lapse imaging of acute cerebral cortical slices of SSLOW-infected Cx3cr1/EGFP mice captured at the early clinical (a), advanced (b, d), and sub-clinical (c) stages of disease. (a) (Figure 4—video 1): An EGFP+ cell (indicated by an arrow) extends a process toward neurons #1 and #2, migrates along this process, and subsequently envelops both neurons while simultaneously extending processes toward neurons #3, #4, and #5. (b) (Figure 4—video 7): EGFP+ cell #1 migrates toward and surveys a neuron (circled), then departs. Subsequently, a second EGFP+ cell (#2) migrates to and interacts with the same neuron. White and blue curves trace the respective migration paths of the two cells. (c) Upper panels: An EGFP+ cell approaches, envelops, and then retracts from a neuron. Middle panels: A migrating EGFP+ cell (indicated by an arrow) surveys five distinct neurons (circled), interacting simultaneously with neurons #1 and #2, followed by #3 and #4. Lower panels: An EGFP+ cell maintains prolonged contact with a neuron. (d) (Figure 4—video 3): An EGFP+ cell (arrow) migrates across the field, initially extending processes (arrowhead) toward a neuron. The cell body then translocates along these processes, briefly contacts one neuron, and continues movement toward another. All videos were recorded over a 3-hr period with 5-min intervals. Nuclei were visualized using Hoechst staining. Scale bars = 20 μm.
Figure 4—video 1
Time-lapse imaging of acute brain slices from a SSLOW-infected Cx3cr1/EGFP mouse at the early clinical stage of the disease.
EGFP-positive cell #1 (highlighted by a rectangle) extends a process toward neurons #1 and #2, migrates along this process, and subsequently envelops both neurons, while concurrently extending processes toward neurons #3, #4, and #5. Meanwhile, EGFP-positive cell #2 exhibits prolonged envelopment of neuron #6, then initiates simultaneous envelopment of neuron #7, while maintaining contact with neuron #6. Neuronal nuclei were visualized with Hoechst staining. Images were acquired over a 3-hr period at a rate of one frame every 5 min.
Figure 4—video 2
Time-lapse imaging of acute brain slices from a SSLOW-infected Cx3cr1/EGFP mouse at the sub-clinical stage of the disease.
An EGFP-positive cell in a rectangle migrates across the field of view, sequentially surveying five different neurons (marked by circles). Neuronal nuclei were visualized using Hoechst staining. Images were acquired over a 3-hr period at a rate of one frame every 5 min.
Figure 4—video 3
Time-lapse imaging of acute brain slices from a SSLOW-infected Cx3cr1-EGFP mouse at the advanced stage of the disease.
EGFP-positive cells within rectangles #1 and #2 exhibit migration across the field of view, initially extending processes toward neurons, followed by soma translocation along these processes. EGFP-positive cell #1 passes neuron #1 indicated by a circle and then moves to a neuron #2. In parallel, an EGFP-positive cell within rectangle #3 moves toward and establishes prolonged contact with a neuronal cell #3 indicated by a circle. Neuronal nuclei were visualized with Hoechst staining. Images were acquired over a 3-hr period at a rate of one frame every 5 min.
Figure 4—video 4
Time-lapse imaging of acute brain slices from a SSLOW-infected Cx3cr1/EGFP mouse at the advanced stage of the disease.
Two EGFP-positive cells extend multiple processes in various directions, reaching out to several neurons. Subsequently, their somata translocate along some of these processes while others retract. Neuronal nuclei were visualized using Hoechst staining. Images were acquired over a 3-hr period at a rate of one frame every 5 min.
Figure 4—video 5
Time-lapse imaging of acute brain slices from a SSLOW-infected Cx3cr1/EGFP mouse at the sub-clinical stage of the disease.
An EGFP-positive cell in a rectangle migrates across the field of view, sequentially surveying six different neurons (marked by circles). Neuronal nuclei were visualized using Hoechst staining. Images were acquired over a 3-hr period at a rate of one frame every 5 min.
Figure 4—video 6
Time-lapse imaging of acute brain slices from a SSLOW-infected Cx3cr1/EGFP mouse at the early clinical stage of the disease.
EGFP-positive cells (#1–4) exhibit prolonged interactions with individual neuronal somas, indicated by ovals. EGFP-positive cell #2 interacts simultaneously with two neuronal somas. Neuronal nuclei were visualized by Hoechst staining. Images were acquired over a 3-hr period at a rate of one frame every 5 min.
Figure 4—video 7
Time-lapse imaging of acute brain slices from a SSLOW-infected Cx3cr1/EGFP mouse at the advanced stage of the disease.
An EGFP-positive cell within rectangle #1 migrates toward and surveys a neuron (circled as #1), followed by a second EGFP-positive cell from rectangle #2, which also migrates to and interacts with the same neuron. Concurrently, other EGFP-positive cells form prolonged contacts with neurons #2 and #3. Neuronal nuclei were visualized using Hoechst staining. Images were captured over a 3-hr period at a rate of one frame every 5 min.
Figure 5 with 8 supplements
Behavioral patterns of low-mobility reactive myeloid cells.
Time-lapse imaging of acute cerebral cortical slices of SSLOW-infected Cx3cr1/EGFP mice, captured at early clinical (a, b) and advanced (c) stages of disease. (a) An EGFP+ cell exhibits prolonged interactions simultaneously with two neurons. (b) An EGFP+ cell shows sustained envelopment of neuron #1, then initiates simultaneous envelopment of neuron #2 while maintaining contact with neuron #1 and possibly neuron #3. (c) An EGFP+ cell moves toward neurons #1 and #2, partially envelops neuron #1, and fully envelops neuron #2. All videos were recorded over a 3-hr period with 5-min intervals. Nuclei were visualized using Hoechst staining. Scale bars = 20 μm. (d, e) 3D reconstructions from confocal microscopy images of fixed cerebral cortical slices immunostained for myeloid cells (IBA1, red) and neurons (NeuN, green) in WT mice infected with SSLOW via the intraperitoneal route, analyzed at early clinical and advanced disease stages. Scale bar = 25 μm.
Figure 5—video 1
3D reconstruction of time-lapse imaging of acute brain slices from a SSLOW-infected Cx3cr1/EGFP mouse at the advanced stage of the disease.
The video highlights the dynamic nature of neuronal envelopment by EGFP-positive microglia. Cells #1 and #2 each partially envelop individual neuronal somas. Cells #3 and #4 exhibit shuttling behavior between two or three neurons (indicated by red arrows) partially enveloping them at the same time. Cell #5 nearly completely envelops one neuronal soma (indicated by red arrow) while simultaneously extending toward another. Neuronal nuclei were visualized with Hoechst staining. Images were acquired over a 3-hr period at a rate of one frame every 5 min.
Figure 5—video 2
Time-lapse imaging of acute brain slices from a SSLOW-infected Cx3cr1/EGFP mouse at the early clinical stage of the disease.
EGFP-positive cells within the marked rectangles exhibit prolonged contacts with individual neurons. Neuronal nuclei were visualized with Hoechst staining. Images were captured over a 3-hr period at a rate of one frame every 5 min.
Figure 5—video 3
Time-lapse imaging of acute brain slices from a SSLOW-infected Cx3cr1/EGFP mouse at the early clinical stage of the disease.
EGFP-positive cell #1 extends multiple processes in various directions, establishing contact with several neurons. Its soma subsequently translocates along some of these processes while others retract. Simultaneously, EGFP-positive cells #2 and #3 each envelop a neuronal soma. Following near-complete envelopment, the myeloid cells retract from neurons. Neuronal nuclei were visualized with Hoechst staining. Images were acquired over a 3-hr period at a rate of one frame every 5 min.
Figure 5—video 4
Time-lapse imaging of acute brain slices from a SSLOW-infected Cx3cr1/EGFP mouse at the early clinical stage of the disease.
Two EGFP-positive cells are observed fully enveloping individual neurons, highlighted by circles. Neuronal nuclei were visualized with Hoechst staining. Images were acquired over a 3-hr period at a rate of one frame every 5 min.
Figure 5—video 5
Time-lapse imaging of acute brain slices from a SSLOW-infected Cx3cr1/EGFP mouse at the advanced stage of the disease.
An EGFP-positive cell is observed fully enveloping individual neuron, highlighted by an oval. Neuronal nuclei were visualized with Hoechst staining. Images were acquired over a 3-hr period at a rate of one frame every 5 min.
Figure 5—video 6
3D reconstruction of time-lapse imaging of acute brain slices from a SSLOW-infected Cx3cr1/EGFP mouse at the advanced stage of the disease.
The video illustrates the dynamic interactions between EGFP-positive cells and neurons. Cell #1 exhibits partial envelopment of a neuron, while cell #2 demonstrates complete envelopment. Neuronal nuclei were visualized with Hoechst staining. Images were acquired over a 3-hr period at a rate of one frame every 5 min.
Figure 5—video 7
Time-lapse imaging of acute brain slices from a SSLOW-infected Cx3cr1/EGFP mouse at the advanced stage of the disease.
An EGFP-positive cell is observed partially enveloping a neuron with a Calbryte-590-positive signal, as pointed by an arrow. The enveloping event is highlighted by an oval. Neuronal nuclei were visualized with Hoechst staining. Calbryte-590 was used to monitor Ca2+ transients. Images were acquired over a 3-hr period at a rate of one frame every 5 min.
Figure 5—video 8
Time-lapse imaging of acute brain slices from a 22L-infected Cx3cr1/EGFP mouse at the early clinical stage of the disease.
Neuronal nuclei were visualized using Hoechst staining. Images were acquired over a 3-hr period at a rate of one frame every 5 min.
Figure 6
Analysis of cell morphology.
Acute cerebral cortical slices were prepared from normal, non-infected Cx3cr1/EGFP mice and from SSLOW-infected Cx3cr1/EGFP mice at the sub-clinical, early clinical, or advanced stages of the disease. Analysis of cell radius (a), cell area (b), cell perimeter (c), and shape index (d) of high- and low-mobility EGFP+ cells in slices from SSLOW-infected mice at three disease stages and normal mice. The midline of the box-and-whisker plot denotes the median, the + represents the mean, and the ends of the box plot denote the 25th and 75th percentiles. N = 3 animals per group; n = 30–90 cells per group, *p < 0.05, **p < 0.01, ***p < 0.01, ****p < 0.0001, ns – non-significant by non-parametric Kruskal–Wallis test with Dunn’s multiple comparison.
Figure 7 with 4 supplements
Sustained Ca2+ bursts in high-mobility myeloid cells.
Acute cerebral cortical slices were prepared from non-infected Cx3cr1/EGFP mice and from SSLOW-infected Cx3cr1/EGFP mice at the early clinical stage of disease. (a) Quantification of signal intensity of Ca2+ puncta within high- or low-mobility EGFP+ cells. Data from normal, non-infected mice are shown for reference. Sustained Ca2+ bursts were detected using Calbryte-590 AM and averaged per cell over a 3-hr period. N = 3 animals per group; n = 24–28 cells per group. *p < 0.05, **p < 0.01, ns = not significant by non-parametric Kruskal–Wallis test with Dunn’s multiple comparisons. (b) Changes in Ca2+ signal intensity in individual EGFP+ cells over the 3-hr imaging session. Images were acquired at 5-min intervals. (c) Time-lapse imaging of acute brain slice from a SSLOW-infected Cx3cr1/EGFP mouse recorded over 3-hr. Upper panels (Figure 7—video 2): EGFP+ cell (#1) envelops a neuronal soma and exhibits low Ca2+ activity. Lower panels: highly mobile EGFP+ cell (#2) displays sustained somatic Ca2+ bursts. Scale bars = 50 μm.
Figure 7—video 1
Time-lapse imaging of acute brain slices from a SSLOW-infected Cx3cr1/EGFP mouse at the advanced stage of the disease.
EGFP-positive cells with high mobility display sustained calcium bursts in their soma or processes. Neuronal nuclei were visualized with Hoechst staining, and Calbryte-590 was used to monitor sustained intracellular Ca2+ activity. Images were acquired over a 3-hr period at a rate of one frame every 5 min.
Figure 7—video 2
Time-lapse imaging of acute brain slices from a SSLOW-infected Cx3cr1/EGFP mouse at the advanced stage of the disease.
EGFP-positive cells with high mobility display sustained calcium bursts in their soma or processes. A circle highlights an EGFP-positive cell that envelops a neuronal soma and displays low Ca2+ activity. Neuronal nuclei were visualized with Hoechst staining, and Calbryte-590 was used to monitor sustained intracellular Ca2+ activity. Images were acquired over a 3-hr period at a rate of one frame every 5 min.
Figure 7—video 3
Time-lapse imaging of acute brain slices from a SSLOW-infected Cx3cr1/EGFP mouse at the advanced stage of the disease.
EGFP-positive cells with high mobility display sustained calcium bursts in their soma or processes. Neuronal nuclei were visualized with Hoechst staining, and Calbryte-590 was used to monitor sustained intracellular Ca2+ activity. Images were acquired over a 3-hr period at a rate of one frame every 5 min.
Figure 7—video 4
Time-lapse imaging of acute brain slices from a SSLOW-infected Cx3cr1/EGFP mouse at the advanced stage of the disease.
Ovals highlight EGFP-positive cells that envelop neuronal somas and display Ca2+ activity. Neuronal nuclei were visualized with Hoechst staining, and Calbryte-590 was used to monitor sustained intracellular Ca2+ activity. Images were acquired over a 3-hr period at a rate of one frame every 5 min.
Figure 8 with 5 supplements
Inhibition of the P2Y6 receptor reduces motility of reactive myeloid cells.
Acute cerebral cortical slices were prepared from non-infected Cx3cr1/EGFP mice and from SSLOW-infected Cx3cr1/EGFP mice at the early clinical stage of disease. (a) Quantification of mean speand total distance traveled by individual EGFP+ cells over a 3-hr period in brain slices from SSLOW-infected mice treated with either MRS-2578 (2 μM) or vehicle control. Data from normal, non-infected mice are shown for reference. N = 3 animals per group; n = 100–250 cells per group. **p < 0.001, ***p < 0.0001 by Tukey’s multiple comparisons test. (b) Time-lapse imaging of MRS-2578-treated acute brain slice from a SSLOW-infected Cx3cr1/EGFP mouse recorded over 3 hr. The EGFP+ cell exhibits bidirectional movement between two neuronal somas (labeled #1 and #2), sequentially enveloping each soma. Scale bars = 20 μm.
Figure 8—figure supplement 1
Gene expression analysis in SSLOW-infected mice.
(a) Heatmap depicting differential gene expression in bulk brain tissue from SSLOW-infected C57BL/6J mice at advanced disease stage. N = 3 mice. (b) Relative gene expression in SSLOW-infected versus age-matched non-infected C57BL/6J mice, normalized by TMEM119 expression. Data are presented as mean ± SD. Statistical significance was assessed using unpaired t-test (*p < 0.05, **p < 0.01, ***p < 0.001). N = 3 mice per group.
Figure 8—figure supplement 2
Envelopment of neurons by P2Y12-positive microglia.
Representative images showing envelopment of neurons by reactive microglia in the cerebral cortex of SSLOW-infected Cx3cr1/EGFP mice (ip inoculation). Brain sections were immunostained with anti-IBA1 (green), anti-P2Y12 (red), and anti-NeuN (gray) antibodies. Right panels present a gallery of envelopment events.
Figure 8—figure supplement 3
Envelopment of neurons by TMEM119-positive microglia.
Representative images showing envelopment of neurons by reactive microglia in the cerebral cortex of SSLOW-infected Cx3cr1/EGFP mice (ip inoculation). Brain sections were immunostained with anti-IBA1 (green), anti-TMEM119 (red), and anti-NeuN (gray) antibodies. Right panels present a gallery of envelopment events.
Figure 8—video 1
Time-lapse imaging of acute brain slices from a SSLOW-infected Cx3cr1/EGFP mouse at the early clinical stage of disease.
EGFP-positive cells exhibit reduced mobility and are observed in close proximity to, or enveloping, neuronal somas (indicated by circles). Slices were treated with 2 μM P2Y6 inhibitor. Neuronal nuclei were visualized with Hoechst staining. Images were captured over a 3-hr period at a rate of one frame every 5 min.
Figure 8—video 2
Time-lapse imaging of acute brain slices from a SSLOW-infected Cx3cr1/EGFP mouse at an early clinical stage of disease.
The EGFP-positive cell highlighted by a rectangle exhibits back-and-forth movement between two neuronal somas, sequentially enveloping each soma. Slices were treated with 2 μM P2Y6 inhibitor. Neuronal nuclei were visualized with Hoechst staining. Images were captured over a 3-hr period at a rate of one frame every 5 min.
Figure 9
In vitro analysis of CD11b+ cell mobility.
CD11b+ cells were acutely isolated from Cx3cr1/EGFP mice at the clinical stage of SSLOW infection or from non-infected Cx3cr1/EGFP mice. Quantification of cell mobility parameters, including mean speed (a), total track distance (b), and mean directional change rate (c) of CD11b+/EGFP+ cells over a 3-hr period, in the presence or absence of N2a cells. In box-and-whisker plots, the midline indicates the median, the ‘x’ denotes the mean, and the box limits represent the 25th and 75th percentiles. N = 9 and 6 animals for non-infected and SSLOW-infected group, respectively. n = 178–354 cells per group. Statistical significance: p < 0.05; p < 0.01; *p < 0.001; **p < 0.0001; ns, not significant by non-parametric Kruskal–Wallis test with Dunn’s multiple comparison. (d) Representative cell tracks of CD11b+/EGFP+ cells over 3 hr. Colored lines indicate trajectories of individual cells.
Tables
Table 1
Sex of animals used in experiments.
| Figure # | Normal | Sub-clinical | Early clinical | Advanced |
|---|---|---|---|---|
| 2, 3, 6 | 2M/1F | 3M | 1M/2F | 2M/1F |
| 7 | 3F | 2M/1F | ||
| Early clinical: MRS-2578 | Mock | |||
| 8 | 2M/1F | 1M/2F | 2M/1F | |
| 9 | 3M/6F | 1M/5F | ||
Table 2
Primer sequences for qRT-PCR.
| Primer | Accession number | Sequence |
|---|---|---|
| P2ry6 | NM_183168 | F 5′-CAGTCTTTGCTGCCACAGGCAT-3’ |
| R 5′-AGCAAGAAGCCGATGACCGTGA-3’ | ||
| P2ry13 | NM_028808 | F 5′-TGGCATCAGGTGGTCAGTCACA-3’ |
| R 5′-TTGTGCCTGCTGTCCTTACTCC-3’ | ||
| P2rx7 | NM_011027 | F 5′-GAACACGGATGAGTCCTTCGTC-3’ |
| R 5′-CAGTGCCGAAAACCAGGATGTC-3’ | ||
| P2rx4 | NM_011026 | F 5′-GCTTTCAGGAGATGGCAGTGGA-3’ |
| R 5′-TGTAGCCAGGAGACACGTTGTG-3’ | ||
| Trem2 | NM_031254 | F 5′-CTACCAGTGTCAGAGTCTCCGA-3’ |
| R 5′-CCTCGAAACTCGATGACTCCTC-3’ | ||
| Tmem119 | NM_146162 | F 5′-ACTACCCATCCTCGTTCCCTGA-3’ |
| R 5′-TAGCAGCCAGAATGTCAGCCTG-3’ | ||
| Tlr2 | NM_011905 | F 5′-ACAGCAAGGTCTTCCTGGTTCC-3’ |
| R 5′-GCTCCCTTACAGGCTGAGTTCT-3’ | ||
| Cd68 | NM_009853 | F 5′-ACTGGTGTAGCCTAGCTGGT-3’ |
| R 5′-CCTTGGGCTATAAGCGGTCC-3’ | ||
| GAPDH | NM_008084 | F 5′-CATCACTGCCACCCAGAAGACTG-3’ |
| R 5′-ATGCCAGTGAGCTTCCCGTTCAG-3’ |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/107650/elife-107650-mdarchecklist1-v1.docx
-
Source data 1
Source data for the Figures containing graphs/plots.
- https://cdn.elifesciences.org/articles/107650/elife-107650-data1-v1.xlsx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Dissecting surveying behavior of reactive microglia under chronic neurodegeneration
eLife 14:RP107650.
https://doi.org/10.7554/eLife.107650.3
