Intravital calcium imaging of meningeal macrophages reveals niche-specific dynamics and aberrant responses to brain hyperexcitability

Simone Carneiro-Nascimento
Chao Wei
Anna Gutterman
Dan Levy author has email address

Department of Anesthesia, Critical Care and Pain Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, United States

https://doi.org/10.7554/eLife.109888.1

Open access
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Figures and data

Imaging meningeal macrophage Ca²⁺ dynamics in awake behaving mice.
(A) Pf4^Cre:GCaMP6s^fl/wt reporter mouse construct for imaging meningeal macrophages Ca²⁺ activity. (B) Experimental procedure for two-photon imaging of meningeal macrophage Ca²⁺ activity. Following the implantation of a headpost and a cranial window, mice were habituated to head restraint and subjected to two-photon microscopy while head-fixed on a running wheel to study meningeal macrophage Ca²⁺ activity. (C) Macrophage Ca²⁺ imaging processing pipeline.

Ca²⁺ dynamic features of meningeal macrophages at steady state.
(A) Left: Mean projection of an example FOV depicting perivascular (P, red; 1, 2) and non-perivascular (NP, blue; 3, 4) meningeal macrophages (white squares). Scale bar 50 μm. Right: Corresponding macrophages with representative 900-second Ca²⁺ activity traces (top) and their fluorescence signal at selected time points (bottom). (B-G) AQuA2-based morphological and Ca²⁺ event functional features of perivascular (P=122 cells) and non-perivascular (NP, n=381 cells) meningeal macrophage. (B) Event area, (C) Event perimeter, (D) Event circularity, (E) Event max DF/F0, (F) Event duration, (G) Event rate. Data (B-G) represent median ± IQR. ***p<0.001, ****p<0.0001, Mann-Whitney U-test. n=37 fields of view (FOVs) from 7 mice.

Intracellular Ca²⁺ signal frequency spectra of macrophage subsets in the steady-state meninges
(A) Left: Clustering of meningeal macrophage Ca²⁺ activity based on frequency-domain features and peak detection. Cluster 1 (purple, n=40) and Cluster 2 (green, n=463). Right: Power spectrum density (PSD) of Ca²⁺ signals for each cluster. (B) Example DF/F0 heatmaps of Cluster 1 and Cluster 2 macrophages. (C-H) Morphological and Ca²⁺ functional features of Cluster 1 and Cluster 2 macrophages. (C) Event area. (D) Event perimeter (E) Event circularity. (F) Event max DF/F0. (G) Event duration. (H) Event rate. (I) Signal-to-noise ratio from Clusters 1 and 2. (J) Distribution of cell types across clusters. Data (C-I) represents median ± IQR. ***p<0.001, ****p < 0.0001, Mann-Whitney U-test Data (J) represents the cell proportion. ****p<0.0001, Chi-square test.

Intracellular propagation and intercellular synchronization of meningeal macrophage Ca²⁺ activity.
(A) Spatial maps of two distinct Ca²⁺ events. Left: Propagating Ca²⁺ activity. Right: Stationary Ca²⁺ activity. (B) Distribution of event propagation profiles in perivascular and non-perivascular meningeal macrophages. (C) Schematic analysis paradigm for detecting synchronous Ca²⁺ activity in meningeal macrophages. (D) Synchronous Ca²⁺ events among meningeal macrophages within a FOV. Left: Mean projection of an example FOV showing Ca²⁺ activity in distinct macrophages (colored/numbered). Scale bar 50 μm. Right: Spatial map of macrophages exhibiting synchronous Ca²⁺ activity. Lines connect macrophages with synchronized Ca²⁺ activity, and colors indicate the extent of Ca²⁺ event synchronization. (E) Distribution of distances across macrophage pairs showing different numbers of synchronous Ca²⁺ events. (F) Linear regression showing poor correlation between distances of macrophage with synchronized Ca²⁺ activity and event delay. (G) Proportion of macrophages exhibiting a specific synchronized interaction (cells interacting only with the same subtype: P-P/NP-NP; cells interacting only with a different subtype: P-NP; mixed (cells interacting with the same and different subtypes).

Ca²⁺ signals of dural perivascular macrophages are functionally coupled to behaviorally driven dural vasomotion.
(A) Experimental paradigm: Locomotion data were acquired during imaging in awake-behaving mice. Behaviorally-evoked changes in meningeal vessel diameter were obtained using segmentation of vessels labeled with a tracer during macrophage Ca²⁺ and further tested for coupling with Ca²⁺ signals. (B) Example data of macrophages with Ca²⁺ activity tuned to locomotion-related dural vessel vasomotion. Locomotion bouts (top trace) and dural vessel diameter (black trace) that was well fit by a GLM (red line) using locomotion state as a predictor. Note vasoconstriction during locomotion, indicating a dural vessel. The two bottom traces depict the Ca²⁺ signal (black traces) of distinct meningeal macrophages that were well-fit by a GLM using dural vessel diameter as a predictor, showing either a negative coefficient (purple) or a positive coefficient (green). (C) Temporal profile of dura vessels GLM coefficients averaged across all well-fitted vessels (n=19). Traces represent the median across all well-fit ROIs, with shaded regions indicating IQR. (D, E) Temporal profiles of GLM coefficient values for macrophages’ Ca²⁺ activity averaged across all well-fitted cells (purple, negative coefficient, n=16; green, positive coefficient, n=13). Traces represent the median across all well-fit macrophages, with shaded regions indicating IQR. (F) The deviance explained (goodness-of-fit estimates) of the vessel diameter data included in the GLM used to predict macrophage Ca²⁺ activity were not statistically different for macrophages showing negative (n=16) and positive (n=13) coefficients, suggesting similar interaction levels. (G) Comparison of peak positive and negative coefficients of well-fit macrophage Ca²⁺ activity/vasomotion GLMs. (H) Center of mass of GLM coefficients indicating that dural vessel diameter changes drive bidirectional changes in fluorescence signal at zero delay. Data (F-H) are median ± IQR. ****p<0.0001 (Mann-Whitney U-test).

Diverse meningeal macrophage Ca²⁺ dynamics following cortical spreading depression (CSD).
(A) Experimental setup and example data: (top) A small burr hole was drilled above the frontal cortex 7 days before Ca²⁺ imaging. Mice were pretreated with saline 30 min before imaging baseline macrophage Ca²⁺ activity (30 min, PreCSD). CSD was then induced with a pin prick, and macrophage Ca²⁺ activity was assessed during CSD (1 min during CSD), and post-CSD (30 min, post-CSD). (Bottom) A raster plot of macrophage Ca²⁺ activity showing the acute and persistent increases and persistent decrease in response to CSD. (B) Example of macrophage Ca²⁺ fluorescence changes following CSD. Images depict the mean projection over the specific experimental timeline. Arrows indicate an increase or a decrease in Ca²⁺ activity. Scale bar 50 μm. (C) Individual responses of perivascular (P, n=21) and non-perivascular (NP, n=32) macrophages showing an acute increase in Ca²⁺ activity. (D) Individual response of P (n=18) and NP (n=37) macrophages exhibiting a persistent increase in Ca²⁺ activity. (E) Individual responses of P (n=32) and NP (n=114) macrophages showing a persistent decrease in Ca²⁺ activity. (F) Proportion of P and NP macrophages showing an acute increase in Ca²⁺ activity or no acute change. (G) Proportion of P and NP macrophages showing a persistent increase, decrease, or no change in Ca²⁺ activity. (H) Proportion of macrophages displaying distinct persistent responses stratified based on their acute response. (I) Baseline (PreCSD) Ca²⁺ activity in macrophages exhibiting persistent increased (n=55) or decreased activity (n=146). Data (C-E) are median ± IQR. ****p<0.0001 (Wilcoxon signed rank test). Data (F-H) represents the proportion of cells, **p<0.01; ****p<0.0001 (Chi-square test). Data (I) are median ± IQR. ****p<0.0001 (Mann-Whitney U-test).

CGRP/RAMP1 signaling mediates CSD-related persistent increase in meningeal macrophage Ca²⁺ activity.
(A) Experimental setup and example data: (top) A small burr hole was drilled above the frontal cortex 7 days before Ca²⁺ imaging. Mice were pretreated with the RAMP1 antagonist BIBN4096 (BIBN) 30 min before imaging baseline macrophage Ca²⁺ activity (30 min, PreCSD). CSD was then induced with a pin prick, and macrophage Ca²⁺ activity was assessed during CSD (1 min during CSD), and post-CSD (30 min, post-CSD). (Bottom) A raster plot of macrophage Ca²⁺ activity showing the acute and persistent increases and persistent decrease in response to CSD. (B) RAMP1 inhibition does not affect the baseline (PreCSD) event rate. Data compared between macrophages imaged in saline-treated mice (n=249 cells) and BIBN-treated mice (n=42 cells). (C) RAMP1 antagonism does not affect the CSD-evoked acute increase in macrophage Ca²⁺. Proportion of macrophages showing an acute response (increase vs. no change) in saline-treated mice (n=249 cells) and BIBN-treated mice (n=42 cells). (D) RAMP1 antagonism does not affect the magnitude of the acute macrophage Ca²⁺ signal. Event max DF/F0 in macrophages showing an acute Ca²⁺ increase in saline-treated mice (n=53 cells) and BIBN-treated mice (n=8 cells). (E) RAMP1 antagonism distinctly inhibits the persistent increase in macrophage Ca²⁺ activity post CSD. Proportion of macrophages showing persistent increase, persistent decrease, or no persistent change in saline-treated mice (n=249 cells) and BIBN-treated mice (n=42 cells). (F) Data (B, C, E) represents the proportion of cells. *p<0.05, Fisher’s exact test. Data (D) are median ± IQR.

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