Multiple sclerosis (MS) patients exhibit neuropsychological symptoms in early disease despite the immune attack occurring predominantly in white matter and spinal cord. It is unclear why neurodegeneration may start early in the disease and is prominent in later stages. We assessed cortical microcircuit activity by employing spiking-specific two-photon Ca2+ imaging in proteolipid protein-immunized relapsing-remitting SJL/J mice in vivo. We identified the emergence of hyperactive cortical neurons in remission only, independent of direct immune-mediated damage and paralleled by elevated anxiety. High levels of neuronal activity were accompanied by increased caspase-3 expression. Cortical TNFα expression was mainly increased by excitatory neurons in remission; blockade with intraventricular infliximab restored AMPA spontaneous excitatory postsynaptic current frequencies, completely recovered normal neuronal network activity patterns and alleviated elevated anxiety. This suggests a dysregulation of cortical networks attempting to achieve functional compensation by synaptic plasticity mechanisms, indicating a link between immune attack and early start of neurodegeneration.
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The data that support the findings of this study are available from the corresponding authors upon request.
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This study was supported by the German Research Council (DFG, CRC-TR-128 (to F.Z., S.B., T.K. and A.S.) and CRC-1080 (to A.S., T.M. and F.Z.)). We thank C. Fois, J. Doering, P.-H. Prouvot, M. Schwalm, E. Witsch, F. Aedo-Jury, K. Radyushkin and K. Robohm for support. We would like to thank A. Zymny, C. Liefländer, H. Ehrengard, O. Levai and I. Gravenitz for excellent technical assistance and D. O’Neill, C. Ernest and S. Stroh for proofreading the manuscript.
The authors declare no competing interests.
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Integrated supplementary information
(a) Cortical neurons stained with Oregon Green BAPTA-1 AM (OGB-1) using multi-cell bolus loading technique. (b) Simultaneously recorded astrocytes counterstained with Sulforhodamine-101 (SR101). (c) An overlay of OGB-1-loaded neurons (green) and astrocytes with SR101 (yellow). Experiment was repeated three times. Scale bar = 25 µm.
Coronal slices from remission animals were stained with the following antibodies to label specific cell types for idenfication of the source of TNFα: (a) astrocytic anti-EAAT1, (b) microglial anti-Iba-1, (c) neuronal marker anti-NeuN, (d) anti-GAD67 for GABAergic neurons and (e) anti-CamKII for CamKII+ excitatory neurons; DAPI was used for nuclear staining. (Center) TNFα was stained with anti-TNFα (green), (Right) Merged images of respective stainings are shown. (Scale bars = 30 µm; arrow heads indicate cells which do not express TNFα, arrows indicate cells which are positive for TNFα.) (f) (Left) Magnification of staining for TNFα, scale bar = 1 µm. (Right) Superresolution image (resolution 36 nm) showing the intracellular compartment, which was delineated by CamKII staining (red). Remarkably, TNF alpha (green) immunosignal was present in the intracellular compartment as well as in the extracellular compartment, which is in line with neuronal TNFα secretion. Representative images from 5 animals, scale bar = 500 nm.
Coronal images of brain slices stained with the following antibodies: (a) anti-NeuN, (b) anti-GAD67, (c) anti-CamKII and DAPI. Note there was less co-expression of TNFα found in anti-NeuN and anti-CamKII staining compared to the EAE-remission mice. (Scale bars = 8 µm; n = 5 mice). (d–g) In-situ hybridization for TNFα in control animals. Stainings for GFAP and neurotrace in sagittal slices. Representative images from 5 animals. (Scale bars overview = 50 µm, inserts = 8 µm).
Supplementary Figure 4 Immunohistochemistry staining for amyloid precursor protein (APP) in remission.
Anti-APP staining in coronal brain slices from remission animals shows no axonal degeneration in the cortex. Representative images from 4 animals. Scale bars overview image = 400 μm, inserts = 50 μm.
Cumulative non-binned normalized frequency graph of healthy controls, relapse, and remission mice; a significant shift in frequency distribution was found in remission compared to control and relapse (mean is displayed, two-sided K-S, P < 0.001) but not in infliximab-treated animals in remission. Control n = 567 neurons, 6 mice; relapse n = 595 neurons, 6 mice; remission n = 650 neurons, 5 mice, infliximab-treated remission n = 525 neurons, 4 mice.
Supplementary Figure 6 Immunohistochemical staining for myelin-basic-protein (MBP) to assess myelin sheath damage in control, relapse and remission.
(a–c) Anti-MBP staining of sagittal slices of the cortex shows intact myelination in relapse and remission animals, similar to control. The lower images are enlargements of the areas enclosed by squares in the respective upper images. Representative images from 4 animals per group. (Scale bars overview = 500 µm, middle and lower inserts = 100 µm, 50 µm respectively).
(a–c) Histological stainings for Mac-3+ activated macrophages and CD3+ T cells in the brain and spinal cord. No Mac-3+ or CD3+ cells were found in control animals but a few Mac-3+ and CD3+ cells could be visualized mainly in the spinal cord of relapse and remission animals. Scale bars of overview images of brain slices = 400 µm, and inserts = 100 µm; scale bars of overview images of spinal cord slices = 400 µm, and inserts = 50 µm. (d) Quantitative assessment of percentage of Mac-3+ activated macrophages in the cortex, including all cortical layers, revealed a significant increase in Mac-3+ macrophage infiltration only in relapse phase (one-way ANOVA, *P = 0.02), not in remission (left panel). Restricting the analysis to layer II/III only resulted in no significant differences between all three groups (right panel). Control n = 16 cortical slices from 4 animals, relapse n = 14 cortical slices from 4 animals, remission n = 18 cortical slices from 6 animals. (e) Analyzing for CD3+ cells in the whole cortex, no significant differences were observed between all groups, albeit a trend towards higher number in relapse (left panel), and a significant increase in relapse when analyzing only layer II/III (right panel; one-way ANOVA, *P = 0.013). No significant differences were observed in remission for either the whole cortex or only layer II/III. Control n = 10 cortical slices from 4 animals, relapse n = 8 cortical slices from 4 animals, remission n = 12 cortical slices from 4 animals. Data represented as mean and SEM.
(a) Anti-Iba-1 staining for microglial morphology in the visual cortex showed no obvious changes in any group, scale for overview images = 50 µm. (b) Quantification of ramification of microglia revealed no significant differences for shape factor, form factor or solidity (one-way ANOVA) between groups. Control n = 6 cortical slices from 3 animals, relapse n = 6 cortical slices from 3 animals and remission n = 6 cortical slices from 3 animals, box-and-whisker plot indicates the median value (center line), the 25–75th percentiles (box) and the 10–90th percentiles (whiskers).
Exemplary immunohistochemical stainings for Nav1.6 or Kv7.3 and βIII-tubulin (βIII-tub) in the visual cortex of (a) control and (b) SJL-EAE remission animals show no changes of expression patterns. Representative images from 4 animals per group.
Supplementary Figure 10 Remission phase mice show normal visual discrimination and rotarod performance.
(a) Schematic illustration of visual discrimination task set-up. (b) Pre-training of EAE mice to perform visual discrimination tasks. No difference observed between healthy controls and mice before EAE induction when pre-training was performed. (c) Time course of the correct choice in visual discrimination task in relation to the clinical EAE score during remission (mean ± SEM; healthy controls n = 10; remission phase animals n = 10). (d) Rotarod behavior task. Latencies (s) until the mice fell off for each group are shown. Box-and-whisker plot indicates the median value (center line), the 25 –75th percentiles (box) and the 10–90th percentiles (whiskers); (two-sided t-test, P = 0.8062; healthy controls n = 9; remission phase animals n = 10).
(a) qPCR analysis for TNFα of primary cortical neurons stimulated with LPS for 24 h, values are normalized to control (Stim. 1, n = 8; Stim. 2, n = 8; and n = 6 for controls, horizontal line depicts median value, Kruskal-Wallis test, **P < 0.01). (b) Quantitative comparison of TNFα release from primary cortical neurons in controls (unstimulated, n = 16), treated with Stim. 1 (n = 14) and Stim. 2 (n = 20) for 24 h. Data points are normalized to control, median value is indicated, Kruskal-Wallis test, *P < 0.05 and ***P < 0.001. Stim. 1: 0.01-0.1 µg/ml LPS, Stim. 2: 1-10 µg/ml LPS; LPS = lipopolysaccharide.
(a-c) Confocal images of brain slices stained with the following antibodies: (a) anti-Iba-1, (b) anti-GFAP, (c) anti-CamKII and DAPI. Astrocytes did not show colocalization with MHC class I (arrow heads). Neurons and microglia showed staining with MHC I (arrows). Representative images from 4 animals. (Scale bars overview = 50 µm, inserts = 8 µm).
We did not observe immune cell infiltration or demyelination in the cortex in SJL remission (a). We could demonstrate that mainly excitatory neurons release TNFα (b) and that the spontaneous neuronal network activity increases (c), which might lead to cell death in cells with highest activity (d). TNFα blocking on the other hand ameliorates neuronal activity and behaviour (e).
Supplementary Figures 1–13-
Real-time video; recording at 30 Hz of a local microcircuit.
Supplementary Video 2 - Two-photon Ca2+ imaging in visual cortex, layer II/III of an EAE mouse, Relapse.
Real-time video; recording at 30 Hz of a local microcircuit.
Supplementary Video 3 - Two-photon Ca2+ imaging in visual cortex, layer II/III of an EAE mouse, Remission.
Real-time video; recording at 30 Hz of a local microcircuit.
Visual discrimination task for mice as depicted in Supplementary Figure 10.
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Ellwardt, E., Pramanik, G., Luchtman, D. et al. Maladaptive cortical hyperactivity upon recovery from experimental autoimmune encephalomyelitis. Nat Neurosci 21, 1392–1403 (2018). https://doi.org/10.1038/s41593-018-0193-2
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