Resting Microglial Cells Are Highly Dynamic Surveillants of Brain Parenchyma in Vivo Axel Nimmerjahn, Frank Kirchhoff, Fritjof Helmchen

This supplement contains:

Materials and Methods
SOM Text
Figs. S1 to S3
References and Notes
Movies S1 to S12

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• Movie S1
  Supplementary Movie S1. Typical motility of resident microglial cells in the intact mouse brain. Each frame is a maximum-intensity projection from stacks of fluorescence images recorded every 45 s (150 – 100 µm below the pia; 2 μm axial spacing). (Image width, 150 μm; rate, 13 fps)
  File Name: NimmerjahnMovS1  (Size: 2.8 Mb)

• Movie S2
  Supplementary Movie S2. Typical microglial cell motility in the resting state imaged through the thinned skull (individual cell). Each image is an overlay of two maximumintensity projection images, recorded at t0 = 0 min and ti = (t0 + i) min. Projection images were created from stacks of fluorescence images acquired during time-lapse recording (1 min sampling interval; 45 – 75 μm below the pia; 1 µm steps). Green and red colors thus indicate new formation and deletion of microglial processes over time, respectively. (Image width, 51 μm; rate, 7 fps)
  File Name: NimmerjahnMovS2 (Size: 1.0 Mb)

• Movie S3
  Supplementary Movie S3. High-resolution time-lapse series showing that microglial cells continually and repeatedly sample the brain parenchyma with highly motile protrusions. Individual images are maximum-intensity projections through stacks of fluorescence images recorded every 15 s with 2-μm axial spacing through the thinned skull. (Image width, 22 μm; rate, 13 fps)
  File Name: NimmerjahnMovS3 (Size: 1.9 Mb)

• Movie S4
  Supplementary Movie S4. Time-lapse series showing spontaneous formation of an inclusion and its transport toward the soma in the normal brain. Images are maximumintensity projections through stacks of fluorescence images recorded every 40 s (2 μm axial spacing; 150 – 110 μm below the pia; every second frame shown). (Image width, 50 μm; rate, 14 fps)
  File Name: NimmerjahnMovS4 (Size: 1.9 Mb)

• Movie S5
  Supplementary Movie S5. Microglial cell protrusions make close contact to neurons and other cortical elements in vivo. The time series is an overlay of the simultaneously recorded green and red fluorescence images of EGFP-expressing microglia and SR101 labeled astrocytes, respectively. Neurons and blood vessels appear as unstained dark areas. In addition, blood vessels are enwrapped by SR101-labeled astrocytic end feet. Images are maximum-intensity projections through stacks of fluorescence images recorded 145 – 125 μm below the pia (2 μm axial spacing; 15 s sampling interval; every second frame shown).(Image width, 54 μm; rate, 13 fps)
  File Name: NimmerjahnMovS5 (Size: 1.3 Mb)

• Movie S6
  Supplementary Movie S6. Enhanced microglia volume surveillance in response to surface application of BCC (50 μM), an ionotropic GABA receptor blocker used to increase neuronal activity. In the time series, BCC application is indicated by a white square in the upper right corner. Each image is a maximum-intensity projection through stacks of fluorescence images (2 μm axial increment) recorded every 40 s in layer 2/3 of mouse neocortex in vivo (every second frame shown). (Image width, 160 µm; rate, 8 fps)
  File Name: NimmerjahnMovS6 (Size: 3.0 Mb)

• Movie S7: Low resolution (5.3 MB)   High resolution (10.0 MB)
  Supplementary Movie S7. Microglial cell activation following targeted BBB disruption of a microvessel using a highly localized laser lesion. Simultaneously recorded green (left) and red (right) channel of EGFP-expressing microglia and SR101 labeled astrocytes, respectively, are shown side by side. BBB disruption is evident in the red channel by local tissue expansion and detachment of astroglial end feet. Immediately after the microlesion (indicated by a white square) nearby microglial cells switch from an undirected surveillance behavior to targeted movement of their processes towards the injured site. Also note the formation and collapse of spherical shaped inclusion in the vicinity of the injured site. The time series was recorded 180 – 135 μm below the pial surface with a 40 s time interval between successive fluorescence image stacks. (One channel image width, 112 μm; rate, 14 fps)
  File Name: NimmerjahnMovS7 (Size: 5.3 Mb)

• Movie S8
  Supplementary Movie S8. Microglia activation following targeted BBB disruption of a microvessel using a highly localized laser-induced microlesion (indicated by a yellow square). Individual images are overlays of the simultaneously recorded green and red fluorescence channel showing EGFP-expressing microglia and Texas Red-dextran labeled blood plasma, respectively. BBB disruption is evident through release of stained blood plasma into the extracellular space, local tissue expansion and gradual staining of damaged BBB components by the released (red fluorescent) dye. Note, that microglial processes immediately invade the affected areas. Phagocytosis is indicated by increased protrusive activity within these areas and inclusion of damaged tissue components. Also note the shielding of nearby microvessel branches by microglial excrescences. The time series was recorded 140 – 80 μm below the pial surface with a 60 s time interval between successive fluorescence image stacks. (Image width, 87 μm; rate, 10 fps)
  File Name: NimmerjahnMovS8 (Size: 4.0 Mb)

• Movie S9
  Supplementary Movie S9. Comprehensive microglia activation following a highly localized laser lesion. Immediately after the microlesion (indicated by a white square) nearby microglial cells switch from an undirected surveillance behavior to targeted movement of their processes towards the injured site. The time series was recorded 130 – 90 μm below the pial surface with a 40 s time interval between successive fluorescence image stacks (every second frame shown). (Image width, 111 μm; rate, 8 fps)
  File Name: NimmerjahnMovS9 (Size: 3.0 Mb)

• Movie S10
  Supplementary Movie S10. Shielding of a microvessel segment by microglial processes following laser-induced microlesion (indicated by a yellow square). The time series is an overlay of the simultaneously recorded green and red fluorescence images of EGFPexpressing microglia and SR101 labeled astrocytes, respectively. Note that only one microglial cell appears to participate in the response to the highly localized injury. Fluorescence image stacks were taken every 30 s between 150 and 110 µm below the pia (every second frame shown). (Image width, 93 μm; rate, 10 fps)
  File Name: NimmerjahnMovS10 (Size: 0.7 Mb)

• Movie S11
  Supplementary Movie S11. Time series showing the formation and collapse of spherical shaped inclusions in the vicinity of a laser-lesioned blood vessel arborization, indicating phagocytosis. Each image is a subvolume projection (2 focal planes; 2 µm apart) of the recorded fluorescence image stacks at the level of microvessel cross section (30 s sampling interval; every fourth frame shown). (Image width, 50 μm; rate, 9 fps)
  File Name: NimmerjahnMovS11 (Size: 2.5 Mb)

• Movie S12
  Supplementary Movie S12. Microglia response to local LPS application. Individual images are overlays of the simultaneously recorded green and red fluorescence channel showing EGFP-expressing microglia and the LPS containing micropipette, respectively. For visualization, 100 µM Alexa Fluor 594 was added to the pipette solution. Two pressure applications are visible through a transient increase in red background fluorescence. In response to LPS application, microglia showed targeted outgrowth of their processes towards the source of inflammation. Note, that the micropipette becomes overgrown by microglia extensions, and that a dense meshwork of processes forms around its tip. Each image is a projection (45 focal planes; 2 µm apart) of the recorded fluorescence image stacks 140 – 50 μm below the pial surface (120 s sampling interval; micropipette tip located at 95 μm below the pial surface). (Image width, 188 μm; rate, 9 fps)
  File Name: NimmerjahnMovS12 (Size: 4.6 Mb)