Microglial reaction towards laser-induced single axon transections within the lateral column of the spinal cord during SOD1^G93A disease course. A, Left images were taken 3 min after axonal transection (autofluorescence and arrow) in control and mutant [preclinical 60-day-old, 90-day-old (onset of disease) and advanced clinical 120-day-old] mice. Images on the right were taken 60 min post lesion. See also Videos S1, S2, S3, S4, S5. B,C, Quantification of the velocity of microglial process movements and of microglial lesion response (increase of EGFP fluorescence in a defined area around the injury) to the injured site from preclinical stages (60-day-old and 75-day-old), onset of disease (90-day-old) to clinical and advanced clinical stages (105-day-old and 120-day-old). Control mice were of corresponding age (60 to 140 days of age). Values are presented as mean ± SEM; ANOVA followed by Tukey test (*p<0.05, **p<0.01, ***p<0.001).

A, Reflected light overview (left side) of the surgically exposed anatomic region. Central nervous system and proximal peripheral nervous tissue is depicted. A corresponding area, marked by the black box, was then recorded by 2P-LSM. The vessels in the lateral column (LC) of the spinal cord could also be recognized on the 2P-LSM image (marked by arrows). The image shows central axons and microglia of the lateral spinal cord as well as spinal root axons and spinal root macrophages. In an advanced clinical stage (120-day-old), axonal degeneration in the LC and in the recorded ventral root (VR), indicated by interruptions and swellings (some exemplary marked by arrows), were observed while dorsal root axons remain unaffected. B, Increase of motor axon degeneration in the lumbar ventral roots from preclinical stages (60-day-old and 75-day-old), onset of disease (90-day-old) to clinical stages (105-day-old and 120-day-old). During this time course, macrophages changed their morphology forming foamy cytoplasm (some exemplary cells are marked by arrows). C,D, Quantification of axonal signal density and cellular area of macrophages during the same disease stages that were shown in B. Control mice were of corresponding age (60 to 140 days of age). E, No morphological reaction of adjacent macrophages towards a laser-induced single axon transection (autofluorescence in green and arrow) was observed in the ventral root of preclinical (75-day-old) mice. The transection led to an acute axonal degeneration at both sides of the injury. Values are presented as mean ± SEM; ANOVA followed by Tukey test (*p<0.05, **p<0.01, ***p<0.001). LC, lateral column; DRL2-L4, dorsal root of the spinal segments L2-L4; VRL2-L3, ventral root of the spinal segments L2-L3; DRGL2, dorsal root ganglion of the spinal segment L2.

Exemplary motor axons and NMJs of musculus tenuissimus (containing mainly slow twitch fibers) were imaged in control and SOD1^G93A (mSOD1) mice. EYFP fluorescence is depicted with a green color table. The vascular network, labeled by central venous application of the dextran Texas Red, is depicted with a red color table. A,B, Control NMJs and the adjacent vascular network are shown in 3 dimensions (in A the same region from 4 different perspectives is depicted). The inserts in B are a maximum intensity projection (MIP, upper insert) and an epifluorescence image (lower insert) of the two NMJs. C, Respective motor axons of a complex of NMJs in a control mouse. Two adjacent NMJs of different morphology are shown in the insert. D, Degeneration of NMJs and their respective motor axons during the course of SOD1^G93A disease in musculus tenuissimus. 60-day-old: morphologically unchanged NMJs when compared to controls; two intact synapses are shown in the inserts. 80-day-old: degenerated (arrowhead) or missing synapses (arrow) at the end of some motor axons; interrupted axon terminals (arrowheads) are shown in the insert. 100-day-old: advanced degeneration of the recorded axon complexes (including the inserts), indicated by missing synapses (arrowhead on the right side of the overview and arrowheads in the right insert), structures of broad-spectrum fluorescence (arrowhead on the left side of the overview and arrowheads in the left insert) and aberrant growth of axons. 120-day-old: interruption of a motor axon (arrowheads) and structures of broad-spectrum fluorescence (arrow, arrowhead in the insert). Note that musculus tenuissimus is innervated mainly by slow-motor neurons. These motor neurons show signs of NMJ denervation in SOD1^G93A mice at approximately onset of disease. Scale bars, 20 µm; inserts, 10 µm.

Spinal cord microglia cells were recorded, shown in part with neighbouring axons, within the lateral column during the disease course of SOD1^G93A. The experiments were performed in double transgenic mice expressing EGFP in microglia and EYFP in projection neurons. For better visualization, EYFP fluorescence is depicted with a red color table in all images. All two-photon images are arranged such that rostral is to the left side. A, confocal image of a SMI32-stained cross section of spinal cord. Motor neurons were stained in the ventral part of the lumbar spinal cord. The autofluorescence of a lesion (arrow) induced by a two-photon laser pulse before perfusion of the mouse indicates the area of in vivo imaging in the lateral column of the spinal cord. B, Time course of axonal degeneration in SOD1^G93A mice. Recordings were obtained in preclinical (75-day-old) and advanced clinical (120-day-old) stages in comparable regions of the lateral spinal cord. In 120-day-old mutant mice axons were mostly swollen and interrupted (some exemplary axons are marked by arrows). Note the green fluorescence of monocytes inside the vessels (vessel boundaries marked by arrowheads). C,D, Quantification of axonal and microglial signal density, respectively, in comparable regions of the lateral spinal cord during the course of disease from preclinical stage (60-day-old), onset of disease (90-day-old) to advanced clinical stage (120-day-old). E, Continuous change of microglial morphology towards a more ovoid and a less ramified shape from preclinical stages (60-day-old and 75-day-old), onset of disease (90-day-old) to clinical and advanced clinical stages (105-day-old and 120-day-old). F,G, Quantification of microglial ramification and process motility during the same stages of disease as shown in E. H, An exemplary microglia cell cluster is depicted in a clinical stage (105-day-old). Values are presented as mean ± SEM; statistical significance determined by using ANOVA followed by Tukey test (*p<0.05, **p<0.01, ***p<0.001). GM, grey matter; WM, white matter; DC, dorsal column; LC, lateral column; DR, dorsal root; VR, ventral root.

Microglial reaction towards laser-induced single axon transections within the spinal cord dorsal column (superficial layers as a sensory part, carrying primarily ascending fibers). A, Left images were taken 3 min after axonal transection (autofluorescence and arrow) in control and mutant (preclinical 60-day-old and advanced clinical 120-day-old) mice. Respective images (right) were taken 60 min post lesion. See also Video S7, S8, S9. B, Quantification of microglial response to the injured site. No significant differences were observed between control and mSOD1 mice. Control mice were of corresponding age (60 to 140 days of age). Values are presented as mean ± SEM; ANOVA followed by Tukey test.

A, Exemplary simultaneous 2P-LSM recordings of axons and macrophages in ventral and dorsal lumbar roots in wild-type and advanced clinical (120-day-old) mice. Degeneration of motor axons in the ventral root and increase of the cellular volume of macrophages in the ventral and dorsal roots were observed in the mutant mouse. B, Distinct degeneration of peripheral axons in a 120-day-old SOD1^G93A mouse (clinical stage), shown by simultaneous recording of the 3 major nerves originating from the sciatic nerve. The tibial nerve was more affected than the common peroneal nerve or the sural nerve. Degenerated axons were abundantly surrounded by foamy macrophages. The imaged region is indicated in the insert (CCD camera image). Note that EYFP fluorescence is depicted with a red color table. DRL2, dorsal root of the spinal segment L2; VRL3, ventral root of the spinal segment L3.