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Deficient neuron-microglia signaling results in impaired functional brain connectivity and social behavior

Abstract

Microglia are phagocytic cells that infiltrate the brain during development and have a role in the elimination of synapses during brain maturation. Changes in microglial morphology and gene expression have been associated with neurodevelopmental disorders. However, it remains unknown whether these changes are a primary cause or a secondary consequence of neuronal deficits. Here we tested whether a primary deficit in microglia was sufficient to induce some autism-related behavioral and functional connectivity deficits. Mice lacking the chemokine receptor Cx3cr1 exhibit a transient reduction of microglia during the early postnatal period and a consequent deficit in synaptic pruning. We show that deficient synaptic pruning is associated with weak synaptic transmission, decreased functional brain connectivity, deficits in social interaction and increased repetitive-behavior phenotypes that have been previously associated with autism and other neurodevelopmental and neuropsychiatric disorders. These findings open the possibility that disruptions in microglia-mediated synaptic pruning could contribute to neurodevelopmental and neuropsychiatric disorders.

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Figure 1: Deficient synaptic multiplicity in Cx3cr1KO mice.
Figure 2: Reduced LFP functional connectivity in Cx3cr1KO mice.
Figure 3: Decreased fMRI functional connectivity in Cx3cr1KO mice.
Figure 4: Decreased social interaction in young Cx3cr1KO mice.
Figure 5: Decreased social interaction and blunted functional connectivity modulation in adult Cx3cr1KO mice.

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Acknowledgements

We thank F. Zonfrillo for expert mouse husbandry, I. Charo (University of California San Francisco) and T. Deller (University of Frankfurt) for providing mice, the EMBL Transgenic Facility for help in rederiving mouse lines, the EMBL Microscopy Facility for access to light and electron microscopes, J. Griesbach and M. Al Banchaabouchi for help in videoscoring, A. Jain, J. Gordon and T. Sigurdsson for assistance in establishing in vivo electrophysiology, A. Galbusera for help in performing MRI acquisitions and F. D'Amato and V. Carola for advice in behavioral testing and analysis. This work was supported by funds from EMBL to C.T.G., R.C.P. and Y.Z. (Y.Z. is an EMBL Interdisciplinary Postdocs (EIPOD) fellow), the IIT to A.G., A.B. and F.S., the Italian Ministry of University and Scientific Research PRIN 2009 to D.R. and a Forschungskredit of the University of Zurich to A.L.V.

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Contributions

Y.Z. designed, carried out and analyzed the in vivo electrophysiology experiments. R.C.P. designed, carried out and analyzed all non-electrophysiological behavioral experiments. A.B., F.S. and A.G. designed, carried out and analyzed the fMRI experiments. L.W. designed, carried out and analyzed the light microscopy experiment. G.B. designed and carried out the electron microscopy experiments that were analyzed together with L.W. In vitro electrophysiological experiments were designed, carried out and analyzed by F.P. and D.R. A.L.V. contributed the custom in vivo electrophysiology recording hardware and expertise. C.T.G. conceived the project and wrote the manuscript with input from Y.Z., R.C.P. and D.R.

Corresponding author

Correspondence to Cornelius T Gross.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 In vitro electrophysiology.

(a,b) Representative traces of patch clamp electrophysiological recordings from CA1 hippocampal neurons from wild-type and Cx3cr1KO mice. (c,d) Sample synaptic events recorded from neurons in wild-type and Cx3cr1KO mice. Noise amplitude, estimated as peak-to-peak amplitude in 5 ms segments of 1kHz filtered traces by Clampfit 10 software, was not statistically different in wild-type and Cx3cr1KO mice (t test; WT, 3.7 ± 0.3 pA, n = 14/14/5 cells/slices/mice; KO, 4.0 ± 0.3 pA, n = 14/12/5; P = 0.44). (e,f) Cumulative distributions of sEPSC from the same cells in (a,b). Wild-type mice displayed frequent large events whereas Cx3cr1KO mice showed few. (g) Passive properties of neurons in wild-type and Cx3cr1KO mice. Cell capacitance and resistance values were provided by Clampex 10 acquisition software. Resting potential was estimated from zero current potential -the potential imposed to the cell to zero the pipette offset after the membrane rupture. The mean values reported for all passive properties are not statistically different (t test; cell capacitance: WT, n = 18/16/4; KO, n = 17/14/5, P = 0.65; cell resistance: WT, n = 20/18/5; KO, n = 17/14/5, P = 0.79; resting potential: WT, n = 17/15/5; KO, n = 9/8/5, P = 0.79). (h) Inter-event distributions of mIPSCs in wild-type (black line) and Cx3cr1KO (gray line) littermate mice at PND15 (WT, n = 22/17/4; KO, n = 23/16/5; D = 0.07, P < 0.00001). The frequency of mIPSCs was slightly higher in wild-type compared to Cx3cr1KO neurons (t test;WT, 4.2 ± 0.5 Hz; KO, 3.3 ± 0.2 Hz; P = 0.045).

Supplementary Figure 2 Light and electron microscopy.

(a) Representative image of NF-200-positive axons in CA1 stratum radiatum. The Thy1::GFP transgene labels sparse neuronal dendrites. Blue indicates DAPI nuclear staining. (b) Quantification of the number of NF-200-positive processes did not reveal any significant difference in axonal density between wild-type and Cx3cr1KO littermates (t test; WT: n = 17 sections from 3 animals, KO: n = 16 sections from 3 animals, P = 0.56). (c–h). Serial electron microscope sections showing an example of a multi-synapse bouton (MSB) making excitatory synapses with three neighboring dendritic spines (S1a, S1b, S2) emanating from three distinct dendritic shafts (D1a, D1b, and D2), two of which derive from the same target neuron (D1a, D1b). In addition, a single-synapse bouton (b) is seen making an excitatory synapse with a single dendritic spine (S3) protruding from a fourth dendrite (D3).

Supplementary Figure 3 In vivo electrophysiology.

(a) Representative LFP recordings from right hippocampus (top, HPC) and prelimbic cortex (bottom, PFC). Thirty seconds of continuous recording are shown. (b) Speed of wild-type and Cx3cr1KO littermate mice during 5 minutes habituation to the three-chambered social interaction apparatus. No significant genotype effect on speed was observed. (c) Partition of dorsal hippocampal LFP theta power into epochs of low (0–5 cm/s) and high (5–10 cm/s) speed did not reveal a genotype effect on theta power in either speed range. (d) The genotype effect on PFC–HPC LFP coherence was maintained following partitioning of the data into low (0–5 cm/s) and high (5–10 cm/s) speed ranges. For panels (b–d), WT: n = 7; KO: n = 10. *P < 0.05, **P < 0.01.

Supplementary Figure 4 Location of in vivo recording electrodes.

Representative Nissl-stained brain sections showing electrolytic lesions produced by LFP electrodes placed in (a) dorsal hippocampus (HPC) and (b) pre- limbic cortex (PFC). Summary of lesion sites of animals included in the LFP coherence experiments superimposed on coronal brain atlas sections indicates electrode localization to (c) dorsal hippocampus and (d) pre-limbic/cingulate cortex (+ wild-type, x knockout).

Supplementary Figure 5 Comparable BOLD signal fluctuation and arterial blood pressure in wild-type and Cx3cr1KO mice during rs-fMRI.

(a) Analysis of the standard deviation (SD) of normalized rs-fMRI BOLD time series data in somatosensory regions, a parameter inversely correlated with anesthesia depth in rodents, produced comparable inter-strain values (t test; WT: n = 9; KO: n = 7; P = 0.47), thus arguing against a differential sensitivity to anesthesia of the two genotypes. (b) This was confirmed by a lack of correlation (Pearson correlation; r2 = 0.05, P = 0.41) between mean arterial blood pressure (MABP), an additional sensitive indicator of anesthesia depth, and PFC–HPC connectivity. MABP values for the wild-type and Cx3cr1KO mice were not different (t test; P = 0.16. WT: n = 9, open circles; KO: n = 7, filled circles).

Supplementary Figure 6 Dorsal-ventral gradient of functional connectivity deficits in Cx3cr1KO mice.

(a) Correlation of PFC–HPC fMRI BOLD signal in Cx3cr1KO mice compared to wild-type littermates showed a gradient from dorsal to ventral hippocampus with greater differences seen in ventral compared to dorsal hippocampal regions (WT: n = 9; KO: n = 7). PFC–HPC Functional connectivity as assessed by LFP coherence was decreased to a lesser extent in (b,d) dorsal hippocampus (dHPC) than in (c,e) ventral hippocampus (WT: n = 4; KO: n = 4). *P < 0.05, ** P < 0.01.

Supplementary Figure 7 Resting-state fMRI functional connectivity analysis.

(a) Heat maps showing correlation (z-score) of resting-state fMRI BOLD signal across brain regions viewed in coronal, transverse, and sagittal sections with a seed in pre-limbic cortex (PFC). Note that overall higher correlations are seen in (left) wild-type compared to (right) Cx3cr1KOlittermates. (b) Anatomical location of seed regions (s01–s15) used to construct correlation matrices (s01: Fra – Frontal Association Area, s02: PrL – Pre-limbic Cortex, s03: M1 – Primary Motor Cortex, s04: Acb – Nucleus Accumbens, s05: Ins – Agranular Insular Cortex, s06: SS – Somatosensory Cortex, s07: Amy –Amygdala, s08: PtA – Parietal Association Cortex; s09: dHPC – Dorsal Hippocampus, s10: Re – Nucleus Reuniens, s11: Vis – Visual Cortex, s12: pdHPC – Postero-Dorsal Hippocampus, s13: iHPC – Ventral Hippocampus, s14: TeA – Temporal Association Cortex, s15: vHPC – Postero-Ventral Hippocampus).

Supplementary Figure 8 Social interaction tests and grooming behavior.

(a, nest homing) Juvenile mice were placed into one corner (start) of a novel open arena filled with fresh bedding and with familiar bedding strewn into the opposite corner (nest) and time spent in each corner (corner 1, corner 2) was recorded during three minutes. Subsequently, the mice were briefly removed while wire mesh tubes were placed into two corners as indicated and the mother was placed into one of the tubes. (a, maternal interaction) Time spent sniffing the tubes (empty, mother) was recorded during five minutes. (b) Adult mice were placed into a three-chambered apparatus with wire mesh tubes in the outer compartments for 5 minutes (Habituation). Subsequently, the mice were briefly removed while a juvenile mouse was placed into one of the tubes. Time spent in the chamber (empty, social) was recorded during ten minutes (Test). (c) Duration of self-grooming during 1 minute following a spray of water to the face was similar in wild-type mice and Cx3cr1KO littermates (t test; WT: n = 23; KO: n = 14; P = 0.99).

Supplementary Figure 9 Correlation between LFP coherence and social behavior.

(a) Averaged time-dependent HPC power spectra in (left) wild-type and (right) knockout mice during the three seconds preceding and following exploratory contact with the tube containing a juvenile mouse in the social preference test. Neither genotype demonstrated a significant change in theta power in either (b) HPC or (c) PFC when comparing the pre-stimulus (Pre) and post-stimulus (During) period (paired t-test; HPC, WT: n = 7, t6 = 1.1, P = 0.3; KO: n = 10, t9 =0.52, P = 0.61. PFC, WT: n= 7, t6 = 1.04, P = 0.34; KO: n = 10, t9 = 1.01, P = 0.34). (d) Distribution of the correlation between PFC–HPC coherence in the theta band and social interaction time estimated using bootstrap (1000 resamples) method. The 95% confidence interval was [0.43, 0.87].

Supplementary Figure 10 Model for immature synaptic multiplicity in Cx3cr1KO mice.

During the first phase of synaptogenesis, each afferent excitatory input makes a single synaptic contact with its target. Action potentials passing down an axon lead to neurotransmitter release at a single synapse. Under these conditions, the amplitude of action potential-dependent synaptic responses (sEPSC) is equivalent to non-action potential-dependent synaptic responses (mEPSC) and synaptic inputs have a unitary multiplicity. Upon completion of synaptogenesis, however, a significant fraction of excitatory inputs makes multiple synaptic contacts with its target via multi-synapse boutons (MSBs, wild-type, left). Action potentials passing down an axon induce simultaneous release at multiple synapses leading to a larger sEPSC, while mEPSC amplitudes remain unaltered. In Cx3cr1KO mice microglia are transiently decreased during development and synaptic pruning is impaired. Because synapse elimination is linked to the formation of MSBs, knockout mice form fewer MSBs (right) resulting in immature synaptic multiplicity and weak functional connectivity. Note that, despite divergent multiplicity, the number of axons and spines in the mature circuits of wild-type and Cx3cr1KO mice are similar.

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Zhan, Y., Paolicelli, R., Sforazzini, F. et al. Deficient neuron-microglia signaling results in impaired functional brain connectivity and social behavior. Nat Neurosci 17, 400–406 (2014). https://doi.org/10.1038/nn.3641

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