doi: 10.1016/j.neuron.2020.11.018.
Epub 2020 Dec 14.
Non-canonical glutamate signaling in a genetic model of migraine with aura
Affiliations
- PMID: 33321071
- PMCID: PMC7889497
- DOI: 10.1016/j.neuron.2020.11.018
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Non-canonical glutamate signaling in a genetic model of migraine with aura
Neuron.
.
Abstract
Migraine with aura is a common but poorly understood sensory circuit disorder. Monogenic models allow an opportunity to investigate its mechanisms, including spreading depolarization (SD), the phenomenon underlying migraine aura. Using fluorescent glutamate imaging, we show that awake mice carrying a familial hemiplegic migraine type 2 (FHM2) mutation have slower clearance during sensory processing, as well as previously undescribed spontaneous "plumes" of glutamate. Glutamatergic plumes overlapped anatomically with a reduced density of GLT-1a-positive astrocyte processes and were mimicked in wild-type animals by inhibiting glutamate clearance. Plume pharmacology and plume-like neural Ca2+ events were consistent with action-potential-independent spontaneous glutamate release, suggesting plumes are a consequence of inefficient clearance following synaptic release. Importantly, a rise in basal glutamate and plume frequency predicted the onset of SD in both FHM2 and wild-type mice, providing a novel mechanism in migraine with aura and, by extension, the other neurological disorders where SD occurs.
Keywords: astrocyte; cortical spreading depression; familial hemiplegic migraine; glutamate; glutamate transporter; iGluSnFR; spreading depolarization.
Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.
Conflict of interest statement
Declaration of interests The authors declare no competing interests.
Figures
Slowed glutamate clearance in awake FHM2 mice (A) Fluorescent glutamate imaging was performed in awake, head-fixed mice by using epifluorescence combined with whisker stimulation. (B) Fluorescent glutamate response (left) in the barrel cortex following a 40-ms whisker stimulation (at 0 ms) with quantification (right). (C) Glutamate clearance rates were determined by fitting the decay of the glutamate response with a 2-term exponential equation (black solid line; blue, glutamate fluorescence from a single trial; dashed line, single-term fit). Boxed inset: magnification of the trace to illustrate poor fit of a single-term equation. (D and E) Glutamate clearance kinetics for τfast (D) and τslow (E). Means, two-sample t test; n = 9 mice/group. Trials, 2-sample Kolmogorov-Smirnov; n = 79 trials for WT and 78 for FHM2. Error bars represent SEM. See also Figure S1.
Glutamatergic plumes in awake FHM2 mice (A) Example of a plume (yellow arrowhead) in a FHM2 mouse measured using 2-photon microscopy (2P). Center panels: magnified view of the plume over time (169 ms/panel). AIP, average intensity projection. Raw images with a Gaussian blur (see STAR Methods). Right: maximum intensity projection (MIP) of change in fluorescence (ΔF) over the entire stimulation trial (33.78 s) illustrates amplitude of the plume relative to all other glutamate signaling. (B) Amplitude (Amp.; top; ΔF/F0), duration (middle; s), and diameter (bottom; μm) of n = 590 plumes from 7 FHM2 mice. Red circle, median; vertical line, mean. (C) Example of a second plume with longer duration and larger diameter than that in (A). Panels are successive imaging frames, starting at the top left corner and ending at the bottom right. (A′ and C′) Quantification of the two plumes. Note that plumes occurred during and outside of whisker stimulation (stim.). (A″ and C″) Kymographs illustrate that both plumes started at a central location and expanded with time. Colorbar as in (A) and (C). (D) AIP from a WT (left) and a FHM2 (right) mouse with colored circles overlaid to indicate location and size of plumes, as well as corresponding traces. WT is representative of their general lack of plumes under baseline conditions. (E) Frequency of plumes by genotype (Wilcoxon rank-sum test; n, number of mice in parentheses). For all figures, “box whisker” represents the median (horizontal line), interquartile range (IQR; “box”), 1.5 × IQR (“whiskers”), and >1.5 × IQR (“+”). See also Figure S1, and Videos S1 and S2.
Inefficient glutamate clearance mediates plumes (A) Imaging schematic and AIP with plume overlay show plumes primarily occurred near the surface of the brain in putative cortical L1a. BV, blood vessel. (B) Quantification of plume frequency by cortical layer in FHM2 (ANOVA with Bonferroni-Holm correction). (C) Low magnification of confocal microscopical fields reveals irregular areas of reduced GLT-1a immunofluorescence (ir) within L1a (white arrowheads). Red arrowheads indicate putative GLT-1a+ glia limitans. (D) Highly magnified electron microscopic fields of immunoperoxidase pre-embedded material exhibit a different frequency of GLT-1a+ profiles (characterized by dark electrodense products) in L1a (left) and L2/3 (right). Electron microscopy confirms GLT-1a+ profiles in the glia limitans (red arrowheads). (E) Top: example image shows an asymmetric synapse with an adjacent GLT-1a+ astrocyte process (AsP). Arrowhead, postsynaptic density (PSD); AxT, axon terminal; Den, dendrite. Bottom: the density of GLT-1a+ AsPs was reduced in L1a compared to L2/3, resulting in a reduced total (t) density of GLT-1a+ profiles. n, neuronal. (F) Left: asymmetric synapse without GLT-1a immunoreactivity (GLT-1a negative). Right: L1a had a larger proportion of GLT-1a negative synapses versus L2/3. (G) For synapses containing adjacent GLT-1a+ AsPs, L1a contained a lower proportion of perisynaptic AsPs (peri.; distance was <300 nm from the edge of the active zone [AZ]/PSD) and greater proportion of extrasynaptic AsPs (extra; distance, >300 nm) compared to L2/3 (p = 0.016; Fisher’s test). Arrowheads, edge of the AZ/PSD to the nearest AsP. (H) TFB-TBOA was superfused though a perforated coverslip (dura intact) in awake mice to inhibit glutamate transporter function. (I) AIP with overlay of plumes (left; 10 min) in a WT mouse before and after TFB-TBOA, as well as quantification of plume frequency across 4 WT mice (0.25–1 mM; paired-sample t test). Port, hole in coverslip. (J) TFB-TBOA-induced plume characteristic in WT compared to spontaneous plumes in FHM2 under similar conditions (two-sample t test). (B and J) n, number mice in parentheses. (C–I) WT. (E and F) Mann-Whitney test. Error bars represent SEM. Box and whisker show 1× and 1.5× the IQR, respectively.∗∗p ≤ 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. See also Figures S2 and S3.
Glutamatergic plumes are consistent with action-potential-independent, spontaneous glutamate release from neurons (A) Schematic illustrating compound application through a perforated coverslip. (B) Blocking NaV channels with TTX (30 μM) blocked the whisker-mediated glutamate response. Left: individual trials (gray) and mean (color) from a single mouse. Black bars indicate whisker stimulations (40 ms at 10 Hz for 1 s). Right: mean response amplitude (n = 7 FHM2 mice). (C) In the same mice as (B), TTX did not inhibit the frequency of plumes (right). Left: AIP with overlay of plumes (10 min) from a single mouse. (D) The CaV channel blocker Ni2+ (1 mM) reduced the frequency of spontaneous plumes in vivo, suggesting plumes depend on neuronal synaptic machinery (n = 7 FHM2 mice). Left: AIP with overlay of plumes (5 min). Images cropped for clarity. (E) Preventing vesicular filling with bafilomycin A1 (BafA1; 4 μM) inhibited plume frequency in FHM2 cortical slices (Wilcoxon rank-sum test), confirming plumes depend on vesicular release. (F) Inhibiting NaV channel inactivation with a brief exposure to veratridine (+Verat.; 10 min, 100–150 μM) was sufficient to increase plume frequency in vivo (Wilcoxon rank-sum test; n = 8 FHM2 mice). (G) SERCA inhibitors increase the frequency of spontaneous miniature postsynaptic currents (see Results for references), and thapsigargin (500 uM) increased the average frequency of plumes in FHM2 mice (n = 6 mice; measured during the first 10 min of application). (H–J) Examples of neural Ca2+ plumes observed in the neuropil of L1a in FHM2 mice expressing GCaMP6s under the Thy1 promoter (putative excitatory neurons) with a cocktail of glutamate receptor (GluR) inhibitors (see Results and STAR methods) to isolate putative presynaptic neural Ca2+. (H) Left: AIP of L1a. Image cropped for clarity. Center: magnified panel of images depicting a neural Ca2+ plume over time in raw (top) and ΔF images (bottom) in the presence of GluR inhibitors and plume inducers, thapsigargin and veratridine. A Gaussian blur added to all images. Time stamps correspond to the start of the event. Images taken from the dashed square in the AIP. M1, mouse 1. Right: the MIP of the ΔF image series (top) and quantification (bottom). (I and J) Two additional examples of neural Ca2+ plumes from different mice than that in (H) (M2 and M3) recorded in the presence of GluR inhibitors alone (I) or with the addition of veratridine (J). MIP of the ΔF image series (left) and quantification (right). (B–G) Gray is animal mean, and color is grand mean. (B, C, D, and G) Paired-sample t test, one-tailed. Box and whisker show 1× and 1.5× the IQR, respectively. See also Figures S3–S5 and Video S3.
A rise in plume frequency predicts onset of veratridine-induced SD regardless of genotype (A) Glutamate fluorescence was recorded during SD induction by superfusing veratridine through a single perforation in the coverslip. Direct current (DC) confirmed SD propagation. (B) Veratridine induced plumes and a rise in basal glutamate fluorescence prior to SD induction. Temporal contour lines and plume overlays (colored circles) illustrate the glutamate wavefront and location (as well as size) of plumes prior to and with SD initiation, respectively. The color of the contour lines and the plume overlays correspond with time. Dashed circle, hole in the coverslip. (C) Corresponds with (B). Top: traces of selected plumes leading up to SD. Bottom: SD was measured as a propagating rise in glutamate fluorescence (proximal to induction site) and DC shift (distal). Vertical dotted lines denote the start of SD in both recordings, illustrating propagation of the wave across the cortex. Glutamate quantification over entire field of view (FOV; 918 × 569 μm). (D) The concentration of veratridine that induced SD (threshold) was lower in FHM2 than in WT. (E) Plume frequency rose with increasing concentrations of veratridine leading up to SD onset in individual mice (bottom) and genotype averages (top; repeated-measures two-way ANOVA). (F and G) The rise in plume frequency occurred at lower concentrations of veratridine in FHM2 than in WT (F) and correlated with SD threshold across individual animals (G). (G) contains two overlapping points (0.3 mM plume rise, 0.5 mM SD threshold; “ = ” in figure). (H) The frequency of plumes prior to SD (at relative concentrations of veratridine for individual mice) was similar between the two genotypes. (I) Basal glutamate fluorescence increased inside the induction port prior to SD. Top: example ΔF/F0 image. Bottom: corresponding fluorescence traces; ∗, time of ΔF/F0 image. (J and K) The slope of the rise in basal glutamate fluorescence was steeper in FHM2 than in WT mice (J) and correlated with SD threshold across individual animals (K). Slope was measured up to the SD threshold for individual animals or 750 μM veratridine—the median threshold for WT—for comparison. (L) The level of basal glutamate fluorescence (mean ΔF/F0) at the concentration of veratridine that induced SD in individual mice was comparable in both genotypes. (M–O) Ni2+ (10 mM) inhibited the rise in plume frequency and basal glutamate fluorescence, as well as increased the SD threshold in FHM2 mice. (M) Similar to (E), quantification of the plume frequency with increasing concentrations of veratridine leading up to SD onset in individual mice. Ni2+ blocked or inhibited the rise in plume frequency in 4 out of 6 FHM2 mice. ∗, no SD. (N) Ni2+ inhibited the rise in basal glutamate fluorescence with veratridine, quantified as a decrease in the average slope. (O) Ni2+ increased the concentration of veratridine needed to induce SD (threshold) in FHM2 mice. ∗, no SD. n, number of mice in parentheses. (D, F, H, J, L, N, and O) Wilcoxon rank-sum test (N and O used a one-tailed version). (G and K) Spearman’s Rho. Two mice were excluded from (F) and (G) (1 from each genotype) because a rise in plume frequency did not occur. These mice were included in (H)–(L). Box and whisker show 1× and 1.5× the IQR, respectively. See also Figure S6 and Video S4.
A rise in plume frequency precedes the onset of KCl-induced SD in WT mice (A) KCl induced plumes prior to SD induction in a single WT mouse. Like Figure 5B, plume overlays and temporal contour lines illustrate the location and size of plumes, as well as the glutamate wavefront, respectively. (B) Select traces of plumes from the same animal as in (A). Color corresponds with the colorbar in (A). Multiple plumes are present in some traces, indicating multiple events occurred in the same location. The increase in basal fluorescence over time is due to the rise in glutamate fluorescence ahead of SD initiation (see E and F below). (C) The frequency of plumes with increasing KCl concentrations up to SD induction for individual animals (n = 6 WT mice). (D) Quantification of the plume frequency during baseline (KCl = 3 mM) and the concentration of KCl that induced SD (relative for each animal) (Wilcoxon signed-rank test; n = 6 WT mice). Gray is animal mean, and color is grand mean. Box and whisker show 1× and 1.5× the IQR, respectively. (E) Basal glutamate fluorescence was relatively stable prior to SD induction, despite increasing concentrations of KCl. An example trace (black) from the same animal as in (A) and (B). ∗, imaging artifact due to the exchange of fluids in the imaging well (truncated for illustration). (F) Basal glutamate fluorescence increased as a brief ramp (Glu. fluo. rise) at the SD induction site just prior to SD initiation (region of interest [ROI] #1, blue trace; corresponds with the colored circle in the inset image of E). By contrast, SD propagation induced a sharp rise in glutamate fluorescence, but no ramp (ROI #2, orange trace). Traces start from the application of 275 mM KCl. A representative of n = 6 WT mice (see Results).
Continued glutamate release in the form of glutamatergic plumes following the propagating SD wavefront (A) KCl (1 M) superfused through a small burr hole near lambda induced an SD that propagated into the imaging window, placed over the barrel cortex (using an intact coverslip). (B) ΔF images quantify the increase in glutamate fluorescence with the propagating SD wave in L1 of a WT mouse. Note that glutamatergic plumes do not precede the SD glutamate wavefront. Each time stamp is relative to the first image (0 s). Top left: AIP prior to SD (raw image). The yellow dashed box corresponds to the magnified images in (C). See (B’) for quantification. (C) A glutamatergic plume that occurred during SD depolarization (following the wavefront) in the same animal as in (B). Time stamp corresponds to 0 s in (B). Peak fluorescence of the plume occurred at 17.23 s. For illustrative purposes, the lower range of ΔF was increased from 0 to 200 ΔF to control for increased basal fluorescence during SD. See (C’) for quantification. (D) An example of all SD-induced plumes in L1 (same mouse as in B). Left: a MIP provides a spatial map of all plumes that occurred during SD depolarization. Center: plume ROIs (circles), with a select set (colored circles) highlighted below and quantified to the right. Right: a ΔF kymograph from the entire FOV illustrates the SD wavefront and all SD-associated plumes over time. Note that plumes occurred after the SD wavefront (representative of n = 8 WT and 9 FHM2 mice). For traces of plumes (bottom), the initial rise in fluorescence is the glutamate SD wavefront, and the colored ROI sits directly above the plume. (E) The frequency of plumes during SD depolarization in L1 was significantly higher than in baseline recordings (prior to SD induction) in WT mice (n = 8 mice). (F) Characteristics of SD-induced plumes from WT mice in cortical L1. Red circle, median; red horizontal line, mean. Box and whisker show 1× and 1.5× the IQR, respectively. (G) A histogram (blue) shows the number of plumes/s over time from all WT mice in L1 and illustrates the latency of each plume relative to the peak (0 s) in basal glutamate fluorescence (Basal Glu.; black). The peak of the plume frequency occurred at 18.5 s. Bin width, 1 s. (H) Similar to WT, SD depolarization increased the frequency of plumes in L1 in FHM2 mice compared to baseline recordings (prior to SD induction; n = 9 mice). (I) A histogram of the number of plumes/s over time in FHM2 mice (red) overlaid with WT (blue; same events as in G), shows that plumes persisted for a longer period following the SD wavefront (time = 0 s) in FHM2 mice. Both histograms were normalized to their relative probability. The peak of the plume frequency in FHM2 mice occurred at 20.5 s (see STAR methods). Inset: the cumulative distribution of plumes over time for both genotypes. (J) SD-induced plumes were larger in diameter (left) and had a longer duration (right) in FHM2 mice than in WT. Plots show a cumulative probability (traces), and insets are a histogram of relative probability (prob.). Both histograms use the same x axis as their respective cumulative plots. (E and H) Paired-sample t test, one-tailed. (F and G) n = 405 plumes from 8 WT mice. (I and J) Two-sample Kolmogorov-Smirnov test; n = 264 plumes from 9 FHM2 mice. (B–G) WT. (H) FHM2. See also Figure S7.
A model of glutamatergic plumes (A) Glutamate release during plumes is likely due to Ca2+-mediated vesicular release from neurons, as compounds that inhibit release (Ni2+ and BafA1) reduce the frequency of plumes (pink lines), whereas those that promote release (veratridine and thapsigargin) increase the frequency of plumes (green arrows). Once glutamate is released, the presence of plumes is gated by impaired or inefficient glutamate clearance by astrocytes. Stimuli that depolarize neural membranes (KCl and SD) are sufficient to induce plumes. This model illustrates glutamate release from a single synapse for clarity, although release from multiple synapses may contribute to a single plume (see Discussion). Pre., presynaptic; Post., postsynaptic. (B) The relationship of plumes to SD. Left: an increase in plume frequency precedes veratridine-induced SD in FHM2 and WT mice, as well as KCl-induced SD in WT, at the initiation site. Right: as SD propagates into the surrounding cortex, plumes occur during the depolarization phase, following the wavefront. Additional work is needed to determine whether the mechanisms of plume generation during SD depolarization overlap with those during spontaneous plumes and SD initiation.
Comment in
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Neuronal plumes initiate spreading depolarization, the electrophysiologic event driving migraine and stroke.Neuron. 2021 Feb 17;109(4):563-565. doi: 10.1016/j.neuron.2021.01.024. Neuron. 2021. PMID: 33600751
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