Microglia remodel synapses by presynaptic trogocytosis and spine head filopodia induction

May combine two claims.

Material engulfed from boutons and axons typically ranges between 0.01 and 0.05 μm3 with an average diameter of 253 ± 24 nm.

“Analysis of the size distribution of inclusions revealed that the material being engulfed from boutons and axons typically ranged between 0.01 and 0.05 µm3 (Fig. 3d) with an average diameter of 253 ± 24 nm.”

May combine two claims.

Microglia have 20 double-membrane structures in process of engulfment, many involving axonal shafts (8/20) and a smaller number involving presynaptic boutons (3/20).

“In addition, we found 20 double-membrane structures that appeared to be in the process of engulfment. Many involved axonal shafts (8/20; Fig. 3b, c and Supplementary Movie 3) and a smaller number involved presynaptic boutons (3/20).”

May combine two claims.

Among microglia-spine contacts, the majority are appositions (minor colocalization, 1.9% of spines) and the rest are encapsulations (>70% spine surface contacted, 1.4% of spines).

“The majority of these contacts presented a relatively minor colocalization of spine and microglia fluorescence (apposition, Fig. 1a, c; n = 171, 1.9% of spines), whereas others were characterized by more extensive colocalization as defined by > 70% of the spine surface contacted by microglia (encapsulation, Fig. 1b, c; n = 123, 1.4% of spines).”

May combine two claims.

Microglial inclusions contain presynaptic material: 17 double-membrane inclusion bodies were found, two of which contained putative presynaptic vesicles of 40 nm diameter.

“We found 17 confirmed double-membrane inclusion bodies (Fig. 3c). Two of these contained putative presynaptic vesicles as indicated by their 40 nm diameter (Fig. 3a, c and Supplementary Movie 2), suggesting that presynaptic material is a substrate for elimination by microglia.”

May combine two claims.

Most encapsulations observed by confocal microscopy appear as simple appositions by electron microscopy (3/5), with only one ROI showing >50% of spine surface contacted by microglia.

“However, most encapsulations appeared as simple appositions (3/5) with only one ROI showing > 50% of the spine surface contacted by microglia (Fig. 2b–f).”

The quote only partly states this claim.

Microglia selectively trogocytose (partially phagocytose) presynaptic structures (boutons and axons) in developing mouse hippocampus.

“Our findings define a set of dynamic microglia–synapse interactions, including the selective partial phagocytosis, or trogocytosis (trogo-: nibble), of presynaptic structures and the induction of postsynaptic spine head filopodia by microglia.”

• Also stated in: § Microglia trogocytose presynaptic elements · hedged

  “These findings argue for the specific trogocytosis of presynaptic structures by microglia and suggest that this activity may be oriented indiscriminately toward axons and synaptic boutons rather than selectively targeting the presynaptic active zone.”

May combine two claims.

All contacted spines were still attached to the dendritic shaft via a spine neck, and 3D FIB-SEM reconstructions did not support phagocytic engulfment of postsynaptic material by microglia.

“using cytoplasmic labeling we noted that all contacted spines were still attached to the dendritic shaft via a spine neck and our 3D FIB-SEM reconstructions did not support the phagocytic engulfment of postsynaptic material by microglia.”

May combine two claims.

Even when microglia eliminate most of a synaptic bouton (2/11 events), presynaptic material remains at the initial site, confirming partial elimination (trogocytosis) of presynaptic boutons.

“Notably, even in those cases where microglia were seen to eliminate most of the synaptic bouton (2/11 events), presynaptic material remained at the initial site, confirming that microglia engage in partial elimination, or trogocytosis, of presynaptic boutons.”

May combine two claims.

There is no difference between WT and CR3-KO in latency of elimination (WT: 6.0 ± 1.6, KO: 3.9 ± 1.1, p=0.28) or number of iRFP inclusions in microglia soma (WT: 2.6 ± 0.4, KO: 2.2 ± 0.6, p=0.51).

“There was also no difference in the latency of elimination (WT: 6.0 ± 1.6, KO: 3.9 ± 1.1, p = 0.28, t-test; Fig. 5d) or in the number of iRFP inclusions found in microglia soma (WT: 2.6 ± 0.4, KO: 2.2 ± 0.6 min, p = 0.51, t-test; Fig. 5e).”

May combine two claims.

Vectorial correlation confirmed that spine head filopodia extended toward the microglia process, varying in length from 0.4 to 3.1 μm with an average of 1.5 μm (n=21).

“Vectorial correlation of spine head filopodia and microglia process movement direction confirmed that filopodia extended toward the microglia process, and varied in length from 0.4 to 3.1 µm with an average of 1.5 µm (n = 21 spine head filopodia formations analyzed, Fig. 6h).”

May combine two claims.

The majority of filopodia induced by microglia originate from mature spine heads, confirmed in electron microscopy datasets.

“we observed that the majority of filopodia induced by microglia originate from mature spine heads (Fig. 6), a finding confirmed in our electron microscopy datasets.”

May combine two claims.

Microglia trogocytosis is restricted to presynaptic boutons and axons, with no evidence for elimination of postsynaptic material.

“Importantly, microglia trogocytosis was restricted to presynaptic boutons and axons, with no evidence for elimination of postsynaptic material.”

May combine two claims.

Microglia-induced spine head filopodia formation was frequently followed by relocation of the spine head to the tip of the filopodium and occasionally resulted in a new bouton contact.

“Our time-lapse imaging study revealed that microglia-induced spine head filopodia formation was frequently followed by a relocation of the spine head to the tip of the filopodium and occasionally resulted in a new bouton contact (Fig. 7).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The speed of microglial process extension and retraction events in organotypic cultures is similar and stable over time (1.9 and 1.8 μm/min, respectively).

“The speed of extension and retraction events was similar and stable over time (1.9 and 1.8 µm/min, respectively; Supplementary Fig. 3e, two-way ANOVA, main effect of time: F2, 12 = 3.48, p = 0.06; main effect of direction: F1, 12 = 0.10, p = 0.77, n = 4 cells) and consistent with previous in vivo imaging studies3.”

• cites [3] Nimmerjahn, A., Kirchhoff, F. & Helmchen, F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308, 1314–1318 (2005). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Ten percent of microglia-contacted spines (3/31) disappeared during imaging, and 13% both appeared and disappeared (4/31) and were classified as transient spines.

“Ten percent of microglia-contacted spines (3/31) disappeared during the imaging session (Fig. 6b), and 13% both appeared and disappeared (4/31), and were classified as transient spines.”

May combine two claims.

Microglia do not phagocytose entire synapses; instead they trogocytose (nibble) synaptic structures.

“However, contrary to previous assumptions, we found no evidence for the phagocytosis of entire synapses. Instead, we observed microglia trogocytosis—or nibbling—of synaptic structures.”

May combine two claims.

The majority (9/15) of converging filopodia were spine head filopodia originating from mature, PSD-containing spines; the rest (6/15) were from dendritic shaft structures.

“Consistent with our live-imaging data, the majority (9/15) were spine head filopodia originating from mature, PSD-containing spines (Fig. 7h), whereas the rest were filopodia extending from dendritic shaft structures (6/15; Fig. 7g).”

Probably not a substantial claim: may not pass S3 (load-bearing).

No detectable iRFP signal was seen during 561 nm illumination.

“Notably, no detectable iRFP signal was seen during 561 nm illumination, most likely to be because iRFP is significantly more dim than tdTomato.”

• AssumptionRests on an assumption: The 561 nm laser excites primarily tdTomato (<20% iRFP peak excitation) and the 638 nm laser excites exclusively iRFP.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

iRFP expression was observed in CA3 neurons exclusively as early as 5 days post infection, and expression in CA3-CA1 Schaffer collaterals reached satisfactory expression level 10 days after infection.

“iRFP was observed in CA3 neurons exclusively as early as 5 days post infection, and expression in CA3-CA1 Schaffer collaterals reached satisfactory expression level 10 days after infection.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Microglia initially respond to culturing by retracting processes and assuming an activated phenotype.

“As shown previously, microglia initially respond to culturing by retracting their processes and assuming an activated phenotype33 (Supplementary Fig. 3b).”

• cites [33] Hailer, N. P., Jarhult, J. D. & Nitsch, R. Resting microglial cells in vitro: analysis of morphology and adhesion molecule expression in organotypic hippocampal slice cultures. Glia 18, 319–331 (1996). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Organotypic hippocampal slice cultures undergo key developmental steps similar to in vivo, including synapse maturation.

“Organotypic hippocampal slice cultures are known to undergo key developmental steps similar to those observed in vivo, including synapse maturation28–31, and have been previously used to study ramified microglia function32.”

• cites [28] Bonhoeffer, T. & Yuste, R. Spine motility: phenomenology, mechanisms, and function. Neuron 35, 1019–1027 (2002). sourceNot checked yet

  Not checked in the free run.

• cites [29] Hasegawa, S., Sakuragi, S., Tominaga-Yoshino, K. & Ogura, A. Dendritic spine dynamics leading to spine elimination after repeated inductions of LTD. Sci. Rep. 5, 7707 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [30] Nägerl, U. V., Willig, K. I., Hein, B., Hell, S. W. & Bonhoeffer, T. Live-cell imaging of dendritic spines by STED microscopy. Proc. Natl Acad. Sci. USA 105, 18982–18987 (2008). sourceNot checked yet

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• cites [31] Verkuyl, J. M. & Matus, A. Time-lapse imaging of dendritic spines in vitro. Nat. Protoc. 1, 2399–2405 (2006). sourceNot checked yet

  Not checked in the free run.

• cites [32] Vinet, J. et al. Neuroprotective function for ramified microglia in hippocampal excitotoxicity. J. Neuroinflamm. 9, 27 (2012). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Microglia have a broad role in synaptic remodeling via the trogocytosis of axonal structures, and the induction and reorganization of postsynaptic sites, to achieve appropriate maturation of circuits.

“Instead, they suggest a broad role for microglia in synaptic remodeling via the trogocytosis of axonal structures, and the induction and reorganization of postsynaptic sites, so as to achieve an appropriate maturation of circuits.”

May combine two claims.

During early postnatal development, microglia undergo morphological maturation that matches synaptic maturation and express receptors for neuronal signaling factors upregulated during this period.

“Clues to this activity come from observations that during early postnatal development microglia undergo morphological maturation that matches synaptic maturation4, and that they express receptors for neuronal signaling factors that are upregulated during this period5.”

• cites [4] Dalmau, I., Finsen, B., Zimmer, J., González, B. & Castellano, B. Development of microglia in the postnatal rat hippocampus. Hippocampus 8, 458–474 (1998). sourceNot checked yet

  Not checked in the free run.

• cites [5] Mody, M. et al. Genome-wide gene expression profiles of the developing mouse hippocampus. Proc. Natl Acad. Sci. USA 98, 8862–8867 (2001). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Inhibitory synapses in hippocampus appear to be unaffected by disruptions of neuron-microglia signaling.

“Notably, inhibitory synapses in hippocampus appear to be unaffected by disruptions of neuron–microglia signaling11 (but see ref. 13).”

• cites [11] Zhan, Y. et al. Deficient neuron-microglia signaling results in impaired functional brain connectivity and social behavior. Nat. Neurosci. 17, 400–406 (2014). sourceNot checked yet

  Not checked in the free run.

• cites [13] Cantaut-Belarif, Y. et al. Microglia control the glycinergic but not the GABAergic synapses via prostaglandin E2 in the spinal cord. J. Cell. Biol. 216, 2979–2989 (2017). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Microglia facilitate circuit maturation by a combination of trogocytosis of the axonal compartment and remodeling of postsynaptic sites.

“suggest that microglia facilitate circuit maturation by a combination of trogocytosis of the axonal compartment and the remodeling of postsynaptic sites.”

May combine two claims.

Microglia frequently re-contact the same spine, suggesting that microglia-spine contacts are non-random.

“microglia frequently re-contacted the same spine, suggesting that the contacts were non-random.”

May combine two claims.

Microglia are broadly involved in structural synaptic plasticity and circuit maturation, both by trogocytosis of presynaptic boutons and axons, and by induction of filopodia from postsynaptic sites.

“Together, these data suggest that microglia are broadly involved in structural synaptic plasticity and circuit maturation, both by the trogocytosis of presynaptic boutons and axons, as well as by the induction of filopodia from postsynaptic sites (Supplementary Fig. 6).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Microglia have a complex role in shaping maturing circuits.

“Together, these studies suggest that microglia have a complex role in shaping maturing circuits.”

May combine two claims.

Microglia can induce profound changes in spine morphology, as occasionally the entire spine stretched during microglial contact (2/31).

“Occasionally, we noted stretching of the entire spine during microglial contact, suggesting that the microglia process was able to induce profound changes in spine morphology (2/31; Fig. 6a, f, i).”

May combine two claims.

These observations confirm that spine head filopodia induction might trigger spine switching and potentially the replacement of inefficient with more efficient synapses.

“These observations confirm that spine head filopodia induction might trigger spine switching and potentially the replacement of inefficient with more efficient synapses41,42,53.”

• cites [41] Richards, D. A. et al. Glutamate induces the rapid formation of spine head protrusions in hippocampal slice cultures. Proc. Natl Acad. Sci. USA 102, 6166–6171 (2005). sourceNot checked yet

  Not checked in the free run.

• cites [42] Breton-Provencher, V. et al. Principal cell activity induces spine relocation of adult-born interneurons in the olfactory bulb. Nat. Commun. 7, 12659 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [53] Schätzle, P. et al. Rapid and reversible formation of spine head filopodia in response to muscarinic receptor activation in CA1 pyramidal cells. J. Physiol. 589, 4353–4364 (2011). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

KO mice lacking fractalkine receptor show transient excitatory hyperconnectivity followed by weak synaptic multiplicity and reduced functional brain connectivity in adulthood, suggesting that failure to eliminate synapses at correct developmental time prevents normal strengthening of synaptic connections.

“KO mice lacking fractalkine receptor show transient excitatory hyperconnectivity followed by weak synaptic multiplicity and reduced functional brain connectivity in adulthood11,12, suggesting that a failure to eliminate synapses at the correct developmental time prevents the normal strengthening of synaptic connections.”

• cites [11] Zhan, Y. et al. Deficient neuron-microglia signaling results in impaired functional brain connectivity and social behavior. Nat. Neurosci. 17, 400–406 (2014). sourceNot checked yet

  Not checked in the free run.

• cites [12] Hoshiko, M., Arnoux, I., Avignone, E., Yamamoto, N. & Audinat, E. Deficiency of the microglial receptor CX3CR1 impairs postnatal functional development of thalamocortical synapses in the barrel cortex. J. Neurosci. 32, 15106–15111 (2012). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

None of the disappearing spines were in contact with microglia at the time of disappearance, arguing for a microglia-independent spine elimination process.

“However, none of these spines were in contact with microglia at the time of disappearance, arguing for a microglia-independent spine elimination process.”

Classified as probably not a substantial claim.

Microglia are highly motile in the uninjured brain, continuously extending and retracting processes through extracellular space.

“The recent discovery that microglia are also highly motile in the uninjured brain2,3, continuously extending and retracting processes through the extracellular space, suggests that they may monitor and contribute to synaptic maturation and function.”

• cites [2] Davalos, D. et al. ATP mediates rapid microglial response to local brain injury in vivo. Nat. Neurosci. 8, 752–758 (2005). sourceNot checked yet

  Not checked in the free run.

• cites [3] Nimmerjahn, A., Kirchhoff, F. & Helmchen, F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308, 1314–1318 (2005). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

KO mice lacking complement factors show cortical excitatory hyperconnectivity, supporting a role for microglia in elimination of excess synapses in mammalian neocortex.

“Knockout (KO) mice lacking complement factors show cortical excitatory hyperconnectivity10, supporting a role for microglia in the elimination of excess synapses in the mammalian neocortex.”

• cites [10] Chu, Y. et al. Enhanced synaptic connectivity and epilepsy in C1q knockout mice. Proc. Natl Acad. Sci. USA 107, 7975–7980 (2010). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Filopodia induction by microglia may be a widespread phenomenon and a possible trigger mechanism for the formation of functional synapses.

“Together, these observations argue that filopodia induction by microglia may be a widespread phenomenon and a possible trigger mechanism for the formation of functional synapses.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The architecture of the case where most filopodia shared their dendrite of origin supports the hypothesis that microglia might promote the formation of class I MSBs.

“the architecture of this particular case where most of the filopodia shared their dendrite of origin supports the hypothesis that microglia might promote the formation of class I MSBs.”

Classified as probably not a substantial claim. May combine two claims.

Trogocytosis is a non-apoptotic mechanism for rapid capture of membrane components and differs from phagocytosis, which involves engulfment and elimination of larger cellular structures (>1 μm).

“Trogocytosis has been described in the immune system as a non-apoptotic mechanism for the rapid capture of membrane components and differs from phagocytosis, which involves the engulfment and elimination of larger cellular structures (> 1 µm)17–19.”

• cites [17] Dopfer, E. P., Minguet, S. & Schamel, W. W. A. A new vampire saga: the molecular mechanism of T cell trogocytosis. Immunity 35, 151–153 (2011). sourceNot checked yet

  Not checked in the free run.

• cites [18] Pham, T., Mero, P. & Booth, J. W. Dynamics of macrophage trogocytosis of rituximab-coated B cells. PLoS. One. 6, e14498 (2011). sourceNot checked yet

  Not checked in the free run.

• cites [19] Joly, E. & Hudrisier, D. What is trogocytosis and what is its purpose? Nat. Immunol. 4, 815 (2003). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Microglia can elicit calcium transients and formation of filopodia from dendritic branches, and are required for learning-induced synapse formation.

“At the same time, studies have pointed to a role for microglia in synapse formation in the adult brain, showing that they can elicit calcium transients and the formation of filopodia from dendritic branches15, and that they are required for learning-induced synapse formation16.”

• cites [15] Miyamoto, A. et al. Microglia contact induces synapse formation in developing somatosensory cortex. Nat. Commun. 7, 12540 (2016). sourceNot checked yet

  Not checked in the free run.

• cites [16] Parkhurst, C. N. et al. Microglia promote learning-dependent synapse formation through brain-derived neurotrophic factor. Cell 155, 1596–1609 (2013). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

tdTomato and iRFP were imaged separately by alternating exposure to 561 and 638 nm light sources with a fixed 585 nm long-pass filter detection system.

“This allowed us to image iRFP and tdTomato separately by alternating exposure to 561 and 638 nm light sources with a fixed 585 nm long-pass filter detection system.”

• AssumptionRests on an assumption: The 561 nm laser excites primarily tdTomato (<20% iRFP peak excitation) and the 638 nm laser excites exclusively iRFP.

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

iRFP is significantly more dim than tdTomato.

“Notably, no detectable iRFP signal was seen during 561 nm illumination, most likely to be because iRFP is significantly more dim than tdTomato.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Following incubation at 4 °C for longer than 30 min, microglia soma enlarged, but recovered normal morphology within 2 h.

“it has been reported that following incubation at 4 °C for longer periods of time (> 30 min) microglia soma enlarged, but recovered normal morphology within 2 h64.”

• cites [64] Sugama, S., Takenouchi, T., Fujita, M., Kitani, H. & Hashimoto, M. Cold stress induced morphological microglial activation and increased IL-1beta expression in astroglial cells in rat brain. J. Neuroimmunol. 233, 29–36 (2011). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

The 3D model generated from manual segmentation was matched to the confocal dataset to confirm perfect correlation.

“The segmentation was carried out manually using iMOD software (http://bio3d.colorado.edu/imod/) and 3D model was generated and matched to the confocal dataset to confirm perfect correlation.”

Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

Fiducial features (vasculature and laser etched markings) were used to register the various microscopy modalities.

“This vasculature, which could also be seen by negative contrast in confocal datasets, acted as fiducial features to register the various microscopy modalities (MicroCT, and low- and high-magnification confocal data).”