ArticlePDF Available

Abstract and Figures

Astrocytes are essential contributors to neuronal function. As a consequence, disturbed astrocyte-neuron interactions are involved in the pathophysiology of several neurological disorders, with a strong impact on brain circuits and behavior. Here, we describe altered cortical physiology in a genetic mouse model of familial hemiplegic migraine type 2 (FHM2), with reduced expression of astrocytic Na ⁺ ,K ⁺ -ATPases. We used whole-cell electrophysiology, two-photon microscopy, and astrocyte gene rescue to demonstrate that an impairment in astrocytic glutamate uptake promotes NMDA spike generation in dendrites of cingulate cortex pyramidal neurons and enhances output firing of these neurons. Astrocyte compensation of the defective ATPase in the cingulate cortex rescued glutamate uptake, prevented abnormal NMDA spikes, and reduced sensitivity to cranial pain triggers. Together, our results demonstrate that impaired astrocyte function alters neuronal activity in the cingulate cortex and facilitates migraine-like cranial pain states in a mouse model of migraine.
Content may be subject to copyright.
Romanos et al., Sci. Adv. 2020; 6 : eaaz1584 5 June 2020
SCIENCE ADVANCES | RESEARCH ARTICLE
1 of 13
NEUROSCIENCE
Astrocyte dysfunction increases cortical
dendritic excitability and promotes cranial pain
in familial migraine
Jennifer Romanos1,2, Dietmar Benke1,2, Daniela Pietrobon3,4,
Hanns Ulrich Zeilhofer1,2,5, Mirko Santello1,2*
Astrocytes are essential contributors to neuronal function. As a consequence, disturbed astrocyte-neuron interactions
are involved in the pathophysiology of several neurological disorders, with a strong impact on brain circuits and
behavior. Here, we describe altered cortical physiology in a genetic mouse model of familial hemiplegic migraine
type 2 (FHM2), with reduced expression of astrocytic Na+,K+-ATPases. We used whole-cell electrophysiology,
two-photon microscopy, and astrocyte gene rescue to demonstrate that an impairment in astrocytic glutamate
uptake promotes NMDA spike generation in dendrites of cingulate cortex pyramidal neurons and enhances output
firing of these neurons. Astrocyte compensation of the defective ATPase in the cingulate cortex rescued glutamate
uptake, prevented abnormal NMDA spikes, and reduced sensitivity to cranial pain triggers. Together, our results
demonstrate that impaired astrocyte function alters neuronal activity in the cingulate cortex and facilitates
migraine-like cranial pain states in a mouse model of migraine.
INTRODUCTION
Astrocytes closely interact with neurons and strongly affect their
functions at the synaptic, cellular, and circuit levels (1). Defective
neuron-astrocyte interactions have been implicated in the establish-
ment and development of several neurological disorders (13).
Migraine is an extremely debilitating disease characterized by re-
current unilateral and severe headaches, frequently accompanied
with several other neurological symptoms (4). However, migraine is
much more than an episodic pain disorder. Several findings indeed
suggest that migraine is a disease affecting a large part of the central
nervous system and characterized by a global dysfunction in sensory
information processing and integration, which also occurs between
migraine episodes (interictal period) (5). For example, patients with
migraine exhibit increased cortical responses to noxious and non-
noxious sensory stimuli during the interictal period (6,7). At present,
the cellular mechanisms responsible for these alterations are largely
unknown. Astrocytes have been proposed to play a role in some
inherited forms of migraine, including familial hemiplegic migraine
type 2 (FHM2), an autosomal dominant form of migraine with aura.
FHM2 is caused by mutations in the Atp1a2 gene, which encodes
the 2 subunit of the Na+, K+-dependent adenosine triphosphatase
(ATPase) (2 NKA) (8), an isoform that is almost exclusively ex-
pressed in astrocytes in the adult brain (9). Expression of the 2
NKA is reduced in the heterozygous Atp1a2+/R887 FHM2 knock-in
(KI) mice, which carry an Atp1a2 missense mutation, causing a
complete loss of function of recombinant 2 NKA (8,10). We pre-
viously showed that these FHM2 mice display impaired glutamate
and K+ clearance by astrocytes of the primary somatosensory cortex,
which, in turn, promotes cortical spreading depression (CSD), the
neuronal correlate of the aura symptoms that precede migraine
headache (3).
In this study, we took advantage of the FHM2 KI mouse model
to understand whether and how a mutation in an astrocyte-specific
protein affects neuronal functions in the cingulate cortex (Cg). This
cortical region is crucial for pain processing and displays altered
functionality in patients with migraine (7,1113). We show that
impairment in astrocytic glutamate uptake in this region strongly
enhances cortical dendritic excitability, especially the generation
of N-methyl-d-aspartate (NMDA) spikes in layer 5 (L5) pyramidal
neurons, and enhances their output firing. Moreover, we reveal that
FHM2 mice display increased sensitivity to head pain triggers. Last,
we show that rescuing the disease-causing mutation in astrocytes of
the Cg recovers neuronal function and reduces the pain phenotype.
Our results provide a clear example of how astrocyte dysfunction
produced by a genetic defect affects neuronal activity in the Cg and
affects sensitivity to head pain triggers.
RESULTS
Astrocytic dysfunctions in the Cg of FHM2 mice
The 2 NKA is physically and functionally coupled to glutamate trans-
porters (GluTs) expressed on perisynaptic astrocyte processes (9,14).
The reduced expression of 2 NKA in heterozygous Atp1a2+/R887
mice (FHM2 mice) results in a reduction in glutamate and K+ buffering
capacity of astrocytes in the primary somatosensory cortex (3,10).
To investigate FHM2-associated alterations in glutamate and K+
uptake in the Cg, a brain region crucially involved in pain process-
ing, we performed whole-cell patch-clamp recordings from astro-
cytes in acute cortical slices (Fig.1A). We recorded synaptically evoked
GluT currents (STCs) and K+ uptake currents induced by electrical
stimuli to L1 afferent fibers with an extracellular electrode and
applied different stimulation paradigms (single pulses and pulse
trains at 50 and 100 Hz; Fig.1,AandB). Glutamate uptake was
significantly slower in the Cg of FHM2 mice compared to their
wild-type (WT) littermates, which was reflected by higher STC decay
time constants () [Fig.1,CandD; single pulse: WT decay=3.11±0.15 ms
1Institute of Pharmacology and Toxicology, University of Zurich, CH-8057 Zurich,
Switzerland. 2Neuroscience Center Zurich, University of Zurich and ETH Zurich, CH-8057
Zurich, Switzerland. 3Department of Biomedical Sciences and Padova Neuroscience
Center, University of Padova, 35131 Padova, Italy. 4CNR Institute of Neuroscience,
Via Ugo Bassi 58/B, 35131 Padova, Italy. 5Institute of Pharmaceutical Sciences, ETH
Zurich, CH-8093 Zurich, Switzerland.
*Corresponding author. Email: mirko.santello@pharma.uzh.ch
Copyright © 2020
The Authors, some
rights reserved;
exclusive licensee
American Association
for the Advancement
of Science. No claim to
original U.S. Government
Works. Distributed
under a Creative
Commons Attribution
License 4.0 (CC BY).
on June 5, 2020http://advances.sciencemag.org/Downloaded from
Romanos et al., Sci. Adv. 2020; 6 : eaaz1584 5 June 2020
SCIENCE ADVANCES | RESEARCH ARTICLE
2 of 13
(n=10 cells, N=5 mice) and FHM2 decay=3.69±0.15 ms (n=14,
N=8; *P=0.017); 50 Hz: WT decay=2.81±0.11 ms (n=10) and
FHM2 decay=3.34±0.18 ms (n=13; *P=0.029); 100 Hz: WT
decay=2.54± 0.13 ms (n=10) and FHM2 decay=3.22±0.18 ms
(n=13; **P=0.009)]. The decay kinetics of the K+ currents following
a train of pulses at 100Hz were also significantly slower in the FHM2
mice [Fig.1E; WT decay=1.72±0.08 s (n=7 cells, N=4 mice) and
FHM2 decay=2.25± 0.18 s (n=10, N=5 mice; *P =0.03)]. We
observed no difference in astrocytic resting membrane potential
nor input resistance between the two groups (fig. S1). These data
indicate that in the Cg of adult FHM2 mice, astrocytic uptake of
neuron-derived glutamate and K+ was impaired.
Slower glutamate clearance by astrocytes may lead to prolonged
and increased glutamate levels in the extracellular space. To directly
= 3.49 ms
= 3.71 ms
= 3.81 ms
Single pulse
10 ms
20 pA
= 3.5 ms
Train of pulses
at 50 Hz or 100 Hz
STC (ms)
*P = 0.029 **P = 0.009
*P = 0.01
7
STC (ms)
STC (ms)
K+(s)
*P = 0.03
1
ROI
Stimulation
GFAP.iGluSnFR SR-101
L1 L2/3
GFAP.iGluSnFR: Extrasynaptic glutamate
Decay (ms)
**P = 0.002
A
WT
100 Hz
= 3.53 ms
= 2.75 ms
50 Hz
= 2.81 ms
WT
WT
0
1
2
3
0
2
4
6
0
2
4
6
0
2
4
6
WT
FHM2
WT
WT
STC 11th pulse STC 11th pulse 100 Hz
B
G
E
WT
FHM2
Synaptically activated
glutamate transporter currents (STC)
D
WT
FHM2
AAV.GFAP.iGluSnFr
WT or FHM2 KI mice
F
Unilateral in cingulate cortex
10 × 50 Hz 10 × 100 Hz
C
0
50
100
150
0
50
100
150 **P = 0.006
FHM2
FHM2
FHM2
FHM2
FHM
2
Single pulse
200 ms
Decay (ms)
10 ms
20 pA
From FHM2 KI (FHM2) mice
and WT littermates
SR-101
Cingulate
cortex
Stim.
electrode
Patch pipette
Layer 1
Fig. 1. Aberrant astrocytic glutamate and K+ uptake in the Cg of FHM2 mice. (A) Schematic representation of the experiment. Scale bar, 30 m. (B) Superimposed
representative traces of the inward current evoked in an astrocyte with different stimulation patterns. (C) The decay time of inward currents evoked by single-pulse stim-
ulation is slower in FHM2 mice (red) compared to WT mice (black). (D) The average STC decay times of the last pulse of the trains at 50 (left) and 100 Hz (right) are signifi-
cantly slower in FHM2 mice. Each point represents the STC decay time in one astrocyte. (E) Decay kinetics of the K+ inward current following trains of 100 Hz stimulation
are slower in FHM2 mice. (F) Injection of AAV.GFAP.iGluSnFr unilaterally in the Cg of WT and FHM2 mice. A typical two-photon experiment showing the expression of
iGluSnFr on astrocytes in the Cg (green) and sulforhodamine 101 dye (SR-101; red) is shown. The theta glass electrode for synaptic stimulation is placed in the inner L1,
and glutamate is imaged from an ROI adjacent to the electrode. Scale bar, 40 m. (G) Upon trains of synaptic stimulation (10 × 50 Hz and 10 × 100 Hz), robust and reliable
increases in iGluSnFr emission could be detected. The decay kinetics of the averaged transients are slower in FHM2 mice. Representative traces are the average of at least
five sweeps. Data are means ± SEM. Two-tailed unpaired t test was used.
on June 5, 2020http://advances.sciencemag.org/Downloaded from
Romanos et al., Sci. Adv. 2020; 6 : eaaz1584 5 June 2020
SCIENCE ADVANCES | RESEARCH ARTICLE
3 of 13
test this prediction, we took advantage of the intensity-based glutamate
fluorescent sensor iGluSnFr (15) that we expressed on the extracellular
side of the astrocytic plasma membrane (Fig.1F and see Materials
and Methods). Two-photon imaging of iGluSnFr glutamate signals
allowed the study of the time course of extracellular glutamate follow-
ing synaptic stimulation. Synaptic activity was evoked by focal electrical
stimulation in L1 of the Cg, and glutamate was imaged in a region of
interest (ROI) in the proximity of the stimulation electrode (Fig.1F).
To estimate the speed of glutamate clearance, we fitted the decay of
the averaged evoked glutamate transients (10 trials) with a monoex-
ponential curve (16). We found that synaptically evoked iGluSnFr
transients in the adult Cg were sensitive to minor changes in the
activity of astrocytic GluTs. Accordingly, partial blockade of GluTs
with subsaturating concentrations of threo-beta-Benzyloxyaspartate
(DL-TBOA) (3 M), a concentration that mimics in WT mice the
slowing of STC decay kinetics produced by the FHM2 mutation (3),
increased the decay time constant of extracellular glutamate transients
in WT mice (fig. S2).
We subsequently recorded iGluSnFr signals in the Cg of FHM2
mice and compared it with WT littermates. The iGluSnFr signals
displayed a longer time course following trains of 50 and 100Hz
stimulations in the FHM2 mice, which were 32 and 29% slower,
respectively [Fig.1G; 50 Hz: WT decay= 75.56±3.62 ms (n =13
slices, N=5 mice) and FHM2 decay=100.9±5.33 ms (n=22, N=7;
**P=0.0019); 100 Hz: WT decay = 65.6±3.32 ms (n=14) and FHM2
decay=84.57 ± 4.77 ms (n=22; **P= 0.006)]. Note that we and
others have previously demonstrated that the size and location of
ROIs, stimulation intensity, and sulforhodamine 101 dye (SR-101)
do not influence the iGluSnFr decay kinetics (16,17). Overall, these
experiments demonstrate that in the adult Cg, astrocytes carrying
the W887R 2 NKA mutation responsible for FHM2 display defective
glutamate and K+ buffering capacity upon repetitive synaptic stim-
ulation, which results in temporally prolonged glutamate spillover.
Facilitation of NMDA spike generation in L5 pyramidal
neurons of FHM2 mice
Pharmacological reduction of astrocytic glutamate uptake capacity
in the cortex increases extracellular buildup of glutamate, which
directly affects NMDA receptor activation in L5 pyramidal neurons
(17). In addition, glutamate spillover promotes the occurrence of
NMDA dendritic spikes (18). These spikes are local events (de-
polarizations) caused by the regenerative and voltage-dependent
activation of NMDA receptors in specific dendritic branches and
have been shown to strongly promote pyramidal cell firing invivo
(19). We tested whether the alteration of astrocytic glutamate clearance
in FHM2 mice would affect NMDA spike generation. We evoked
NMDA spikes in the distal dendrites of L5 pyramidal neurons by
focal synaptic stimulation (paired pulse, 50 Hz) in close proximity
to single branches of tuft dendrites in L1 of the Cg (Fig.2A). In-
creasing stimulation intensities caused a nonlinear increase in the
amplitude and area under the curve (AUC) of the second pulse,
which is characteristic of NMDA spikes (20). This nonlinear increase
was abolished in the presence of NMDA receptor blocker d,l-2-
amino-5-phosphonovaleric acid (AP-V) (Fig.2B).
As predicted from the reduced glutamate clearance in FHM2 mice,
these mice displayed facilitated NMDA spike generation reflected
by higher amplitude and AUC of the voltage responses evoked by
L1 synaptic stimulations [Fig.2C; second pulse amplitude: WT,
1.56±0.4 mV (n=12 cells, N=8 mice) and FHM2, 9.13±2.48 mV (n=13,
N=6; ***P<0.0001); second pulse AUC: WT, 40.64±13.9 mV ms
(n=11) and FHM2, 411.5±131 mV ms (n=12; *P<0.05); stimula-
tion intensity, 2 mA]. Partial blockade of GluTs with subsaturating
concentrations of the GluT blocker dl-TBOA (3 M) significantly
enhanced NMDA spike generation in L5 pyramidal neurons in WT
mice (fig. S3), mimicking the effect of the FHM2 genetic mutation.
Dendritic NMDA spikes have been reported to promote somatic
firing in neurons of the somatosensory cortex (19). The main apical
dendrite of L5 pyramidal neurons of the Cg displays a remarkably
low dendrite-to-soma attenuation of slow synaptic inputs compared to
other cortical regions (21), suggesting that NMDA-mediated dendritic
depolarizations may have an even stronger influence on somatic depo-
larization and action potential (AP) firing in this region. We find that
our NMDA spike induction protocol easily triggered somatic firing
(Fig.2D). Consequently, we predicted that the facilitation of NMDA
spikes that we identified in FHM2 Cg L5 pyramidal neurons should
lead to increased firing of these neurons. We found that the probability
of AP firing following NMDA spikes was significantly higher in FHM2
mice compared to their WT littermates (Fig.2D). These data demon-
strate that in the Cg, defective glutamate uptake and the prolonged
presence of synaptically released glutamate are accompanied by a fa-
cilitation in NMDA spikes and somatic firing of L5 pyramidal cells.
We then investigated whether synaptic activity is altered in FHM2
mice. We first recorded miniature excitatory postsynaptic currents
(mEPSCs) from L5 pyramidal cells of WT and FHM2 mice
(Fig.3,A, B, and C) and found no change in baseline synaptic activity
(mEPSC frequency and amplitude) between the two groups [mEPSC
frequency: WT, 3.02± 0.71Hz (n =7 cells) and FHM2 KI,
4.53±1.54Hz (n=9 cells; P=0.43); mEPSC amplitude: WT,
14.81±1.35 pA and FHM2 KI, 13.09±0.82 pA (P=0.27)]. We
additionally recorded AMPA-mediated EPSCs evoked with single-
pulse and paired-pulse extracellular synaptic stimulations (Fig.3,D and E).
Upon single-pulse stimulations, both the amplitude and decay
kinetics of EPSCs were similar in WT and FHM2 mice [amplitude:
WT, −117±18.04 pA (n=7 cells) and FHM2 KI, −118.6±12.03 pA
(n= 6 cells; P=0.94); decay: WT, 17.7 ±2.1 ms (n=7 cells) and
FHM2 KI, 18.29±1.4 ms (n=6 cells; P=0.82)]. Similarly, upon
paired-pulse stimulation at 20 Hz, the second-over-first EPSC
amplitude ratio was comparable in WT and FHM2 mice [second/
first amplitude ratio: WT, 1.73±0.09 (n=7 cells) and FHM2,
1.54±0.08 (n=7 cells; P=0.17)]. These datasets strongly argue
against alterations in glutamate release or in AMPA-mediated
synaptic transmission in the Cg of FHM2 mice.
Local astrocytic defects are responsible for NMDA-mediated
neuronal dysfunctions
Excitatory neuronal activity appears to be highly increased in the
Cg of FHM2 mice. Nevertheless, to what extent the local astrocytic
malfunction is responsible for the modifications in neuronal activity
is not clear. To address this question, we compensated for the astro-
cyte dysfunction in the Cg region of FHM2 mice by expressing the
WT form of 2 NKA (Atp1a2) in astrocytes. To deliver Atp1a2,
we used an adeno-associated virus (AAV) of the 5/2 serotype that
preferentially targets astrocytes and took advantage of the astrocyte
promoter hGFAP (AAV.hGFAP.ATP1A2) (22). As a control, AAV
of the same serotype and with the same vector backbone, but
containing only enhanced green fluorescent protein (eGFP)
(AAV.hGFAP.eGFP), was injected (Fig.4A; also see Materials and
Methods). Since AAV vectors’ size is limited to ~4.7 kb, it was not po ssib le
on June 5, 2020http://advances.sciencemag.org/Downloaded from
Romanos et al., Sci. Adv. 2020; 6 : eaaz1584 5 June 2020
SCIENCE ADVANCES | RESEARCH ARTICLE
4 of 13
to include a reporter gene in the virus with ATP1A2. Therefore, to visual ize
the injection site, we injected a mixture of 0.5l of the two viruses
with a ratio of 2:1 of AAV.hGFAP.ATP1A2 to the control virus
(AAV.hGFAP.eGFP). Two to 3 weeks following injections, immuno-
histochemistry experiments showed that AAV.hGFAP targeted
astrocytes with no apparent neuronal expression (fig. S4). Consistent
with previous findings on other brain regions (10), the Cg of FHM2
mice showed a substantial reduction of 2 NKA expression levels
(about 70%) compared to WT littermates (fig. S5A). This reduction
was significantly, albeit not fully, restored in FHM2 mice injected
with the rescue virus (fig. S5A). The Western blot experiments were
performed 15 days post-injection (d.p.i.) of the viruses. Accordingly,
the functional recovery of the STC decay kinetics was also incomplete
at 15 d.p.i. (fig. S6). This may, at least in part, account for the incomplete
restoration of 2 NKA expression. For this reason, the functional and
behavioral experiments (see later) were performed mostly at 21 d.p.i., wh en
the STC decay kinetics became similar to WT levels (fig. S6). In addition,
it is also likely that the Cg tissue extracted for Western blot analysis con-
tained regions with a mixture of high and low (or no) virus expression.
We additionally evaluated the expression levels of the astrocytic
GluTs, GLT-1 and GLAST, that both play a crucial role in glutamate
uptake in the Cg (17). We observed a 25% reduction of GLAST ex-
pression in the Cg of FHM2 mice, which was restored to WT levels in
FHM2 mice injected with the rescue virus (fig. S5B). On the other
hand, no changes in GLT-1 expression levels were observed between
the different groups (fig. S5C).
We then performed electrophysiological recordings in the same
manner as in Fig.1 from FHM2 astrocytes either expressing WT Atp1a2
or the eGFP control. Our data show that both K+ [Fig.4B; control decay=
2.66±0.16 s (n=9 cells, N=4 mice) and rescue decay=2.14±0.13 s
(n=10, N=4; *P=0.02)] and active glutamate clearance by astrocytes
became significantly faster in FHM2 mice in which WT Atp1a2 was
expressed compared to FHM2 mice that were injected with the control
virus [Fig.4C; single pulse: control decay=3.3±0.06 ms (n=10 cells,
N= 4 mice) and rescue decay= 2.6±0.13 ms (n=12, N= 4;
***P=0.0003); 50 Hz: control decay = 3.24±0.06 ms (n=10) and
rescue decay=2.46±0.18 ms (n=12, **P=0.001); 100 Hz: control
decay = 3.11±0.04 ms (n=10) and rescue decay =2.31± 0.12 ms
(n=12; ***P=0.0001)]. The decay kinetics of the uptake currents in the
rescued FHM2 mice became comparable to those observed in WT mice
(fig. S7A). The astrocytic resting membrane potential was slightly
but significantly hyperpolarized in rescued FHM2 mice compared
to those injected with control virus, with no difference in input
resistance (fig. S8).
AP-V
2nd pulse amplitude (mV)
Stim. intensity (mA)
***
*
Stim. intensity (mA)
2nd pulse area (mV.ms)
*
**
Stim. intensity (mA)
Probability of AP ring
3.5
2 mA
2.5
3
4
–70 mV –72 mV
WT FHM2
50 ms
10 mV
*
***
WT
FHM2
5 mV
20 ms
–73 mV
–72 mV
WT
FHM2
BC
D
WT
FHM2
1.0
1.5
2.0
2.5
0
500
1000
1.0
1.5
2.0
2.5
0
5
10
15
1
1.5 2
2.5 3
3.5 4
4.5 5
5.5
0.0
0.5
1.0
5 mV
20 ms
A
Stimulation
L1
L2-L3
L5
Biocytin
Fig. 2. Facilitation of NMDA spike generation in L5 pyramidal neurons in the Cg of FHM2 mice. (A) Image of whole-cell recording from the soma of a biocytin-labeled
L5 pyramidal cell in the Cg showing the location of the recording pipette (L5) and stimulation electrode (in close proximity to single branches of tuft dendrites in L1 of the
Cg). Scale bar, 50 m. (B) Representative traces of NMDA spikes evoked by focal synaptic stimulation (paired pulse, 50 Hz) of increasing stimulation intensities that cause
an abrupt and nonlinear increase in amplitude and AUC of the second pulse, which is characteristic of NMDA spikes. This nonlinear increase is abolished in the presence
of NMDA receptor blocker AP-V (50 M; box). (C) The amplitude and the AUC of the second pulse are significantly higher in FHM2 mice compared to WT mice upon lower
stimulation intensities. (D) Paired-pulse stimulation had a higher probability to evoke a somatic action potential (AP) in FHM2 mice. Data are means ± SEM. Two-way
analysis of variance (ANOVA) with Bonferroni post hoc test and Z score were used.
on June 5, 2020http://advances.sciencemag.org/Downloaded from
Romanos et al., Sci. Adv. 2020; 6 : eaaz1584 5 June 2020
SCIENCE ADVANCES | RESEARCH ARTICLE
5 of 13
In light of these results, we investigated whether the rescue of the
astrocytic dysfunctions could indeed affect the neuronal defects
observed in FHM2 mice. To this end, we evoked NMDA spikes in the
distal dendrites of L5 pyramidal neurons by focal synaptic stimula-
tion as in previous experiments (Fig.4,DandE). The amplitude and
AUC of NMDA spikes were significantly lower in FHM2 mice in-
jected with WT Atp1a2 compared to FHM2 mice injected with the
control virus [Fig.4F; second pulse amplitude: control, 12.56±2.11 mV
(n=7 cells, N=3 mice) and rescue, 4.8±1.52 mV (n=11, N=4;
***P<0.0001); second pulse AUC: control, 432.7±87.7 mV ms
(n=7) and FHM2, 198.15±69 mV ms (n=12; **P<0.01); stimu-
lation intensity, 2.5 mA]. The values in the rescued FHM2 mice were
comparable to those in WT mice (fig. S7B). This rescue was also
accompanied by a significantly lower probability of AP firing follow-
ing NMDA spikes in the rescued FHM2 mice (Fig.4G).
Local astrocyte dysfunction in the Cg influences orofacial
pain in FHM2 mice
The Cg is a critical cortical region in encoding cephalic pain. Altered
neuronal activity in this brain area has been reported to influence
the activation and sensitization of pain pathways in pathological pain
conditions (23). Whether local astrocyte dysfunction in the Cg can
10 pA
P = 0.43 P = 0.27
Wild type
FHM2 KI
mEPSCs
0
500
1000
1500
2000
2500
0
50
100
150
IEI (ms)
Cumulative frequency (%)
0
20
40
60
0
50
100
150
Amplitude (pA)
0
5
10
15
20
25
Amplitude (pA)
WT
0
5
10
15
20
Frequency (Hz)
WT
150 ms
25 pA
10 ms
WT
KI
WT
KI
Amplitude (pA)
–250
–200
–150
–100
–50
0
Decay time (ms)
0
10
20
30
0.0
0.5
1.0
1.5
2.0
2.5
2nd/1st EPSC
amplitude ratio
P = 0.17
FHM2
WT
FHM2
WT
FHM2
WT
WT FHM2
P = 0.94 P = 0.82
20 ms
50 pA
AMPA-mediated evoked EPSCs
FHM2
FHM2
Cumulative frequency (%)
20 pA
10 ms
WT
Single pulse
FHM2
Paired pulse at 20 Hz
A
B
C
D
E
Fig. 3. Baseline synaptic activity is similar in L5 pyramidal cells of FHM2 and WT mice. (A) Representative example traces (6 s) of whole-cell mEPSCs recordings from
WT mice (left) and FHM2 KI mice (right). Average mEPSCs are shown above the traces. (B) Cumulative frequency plots of interevent intervals (IEI) (left) from WT and FHM2
KI and cumulative frequency plot of mEPSCs amplitudes from the same cells (right) calculated from the presented traces above. (C) No difference is observed in mEPSCs
frequency between WT and FHM2 KI slices. Data points represent individual cells. (D) Left: Example traces of AMPA-mediated evoked EPSCs following a single pulse in WT
(black) and FHM2 mice (red). Right: The amplitude of EPSCs following a single pulse is similar in WT and FHM2 mice. Decay kinetics are also similar in WT and FHM2 KI mice.
(E) Left: Example traces of AMPA-mediated evoked EPSCs following a paired-pulse stimulation at 20 Hz in WT and FHM2 mice. Right: The second-to-first EPSC amplitude
ratio is not different in WT compared to FHM2 mice. Data are means ± SEM. Two-tailed unpaired t test was used.
on June 5, 2020http://advances.sciencemag.org/Downloaded from
Romanos et al., Sci. Adv. 2020; 6 : eaaz1584 5 June 2020
SCIENCE ADVANCES | RESEARCH ARTICLE
6 of 13
100 pA
8 ms
0
1
2
3
4
0
1
2
3
4
0
1
2
3
4
= 3.04 ms
STC (ms)
STC (ms)
STC (ms)
AAV.ATP1A2
100 Hz
AAV.eGFP
K+ (s)
0
1
2
3
4*P = 0.02
***P = 0.0001
**P = 0.001
***P = 0.0003
Single pulse 50 Hz 100 Hz
= 3.3 ms
= 2.12 ms
= 3.22 ms
= 1.93 ms = 1.91 ms
STC 11th pulse STC 11th pulse
AAV.ATP1A2
AAV.eGFP
Stim. intensity (mA)
Stim. intensity (mA)
**
***
*
*******
Probability of AP ring
Stim. intensity (mA)
Stim.
electrode
Patch pipette
Biocytin AAV.GFAP.eGFP
L1 L2
AAV5/2.hGFAP.eGFP (control)
or
AAV5/2.hGFAP. ATP1A2 (rescue)
FHM2 KI mice
21 d.p.i.
AAV.eGFP
AAV.ATP1A2
AB
C
DE F
G
Unilateral in cingulate cortex
AAV.eGFP
AAV.eGFP
AAV.ATP1A2
AAV.eGFP
AAV.ATP1A2
AAV.ATP1A2
1.0
1.5
2.0
2.5
0
5
10
15
20
1.0
1.5
2.0
2.5
0
200
400
600
2nd pulse amplitude (mV)
2nd pulse area (mV.ms)
0.0
0.5
1.0
1
1.5 2
2.5 3
3.5 4
4.5 5
5.5
20 mV
20 ms
1.5 mA
2.5
3.5
3
2
5 mV
10 ms
1 s
AAV.eGFP
AAV.ATP1A2
L1
L2-L3
L5
Stimulation
AAV.eGFP
AAV.ATP1A2
Fig. 4. Local astrocytic defects are responsible for NMDA-mediated neuronal dysfunctions. (A) AAVs containing the WT form of Atp1a2 (AAV5/2.hGFAP.ATP1A2) or
a control virus (AAV5/2.hGFAP.eGFP) is unilaterally injected in the Cg of FHM2 mice. At 21 d.p.i., astrocytes expressing either GFP or Atp1a2 in L1 of the Cg cortex are
targeted for recording in acute brain slices while stimulating nearby neurons. Scale bar, 40 m. (B) Example traces of the K+ current following trains of 100 Hz stimulations
in FHM2 mice injected with the control virus (red) and those injected with the rescue virus (blue). The decay kinetics of K+ currents are significantly faster in rescued FHM2
mice compared to control FHM2 mice. (C) Upon all stimulation patterns (single pulse and trains of 50 and 100 Hz), the decay kinetics of STCs are faster in rescued mice
compared to the control group. (D) Image of whole-cell recording from the soma of a biocytin-labeled L5 pyramidal cell in the Cg surrounded by eGFP-expressing astro-
cytes. Scale bar, 40 m. (E) Representative traces of NMDA spikes evoked by focal synaptic stimulation (paired pulse, 50 Hz) in control FHM2 mice (red) and in rescued
FHM2 mice (blue). (F) Both the amplitude and the AUC of the second pulse are significantly lower in rescued FHM2 mice compared to the control group. (G) Paired-pulse
stimulation had a lower probability to evoke a somatic AP in rescued FHM2 mice (blue) compared to control mice (red). Data are means ± SEM. Two-tailed unpaired t test,
two-way ANOVA with Bonferroni post hoc test, and Z score were used.
on June 5, 2020http://advances.sciencemag.org/Downloaded from
Romanos et al., Sci. Adv. 2020; 6 : eaaz1584 5 June 2020
SCIENCE ADVANCES | RESEARCH ARTICLE
7 of 13
facilitate cranial pain in FHM2 mice is unknown. To explore this
possibility, we activated the cranial pain pathway via a single systemic
injection of the nitric oxide donor nitroglycerin (NTG). NTG is con-
sidered a reliable cranial pain trigger especially in migraine-susceptible
patients. Only in patients with migraine, NTG induces a delayed
migraine-like headache with associated features (e.g., premonitory
symptoms) that resemble their own spontaneous migraine attacks
(24). Systemic injections of NO donors have also been used in rodents
(25) and have been shown to evoke hypersensitivity to touch (typical
migraine symptom), particularly in mice carrying a mutation associated
with migraine with aura (26). To assess the development of facial
mechanical sensitization upon NTG injections, we gently poked the
mice in the orofacial region with von Frey filaments and scored the
evoked nocifensive behavior (orofacial pain score; see Materials and
Methods). This scoring system has been previously used to assess
trigeminal neuropathic pain in rodent models (27). We first found
that NTG [10mg kg−1, intraperitoneally (i.p.)] triggered facial
mechanical hypersensitivity in both WT and FHM2 mice at 30, 60, and
120min post NTG injection compared to mice injected with saline
[Fig.5, Band C; AUC for WT: saline, 67.56± 12.50 (N=6 mice)
and NTG, 165.5±19.08 (N=6; **P=0.0016); AUC for FHM2:
saline, 71.32± 11.72 (N=6) and NTG, 190.3±23.92 (N =6;
**P=0.0012)]. FHM2 mice developed orofacial hypersensitivity upon
NTG doses that were ineffective in WT littermates [5mg kg−1;
Fig.5,DandE; AUC for WT: saline, 91.51±19.11 (N=6 mice) and
NTG, 58.90± 8.88 (N=8; P=0.12); AUC for FHM2: saline,
67.16±20.29 (N=7) and NTG, 146.0±13.13 (N=7; **P=0.0068)].
These results suggest that the FHM2 mutation promotes the develop-
ment of NTG-induced facial mechanical hypersensitivity. The
same phenotype was observed with a lighter von Frey filament of
0.025 g (fig. S9). Activity tests showed no difference between
saline- and NTG-treated mice or between FHM2 and WT litter-
mates, suggesting that the different responses to mechanical
stimulation were not accompanied by alterations in motor func-
tion (fig. S10, A and B).
Since the Cg is implicated in pain signaling and development of
mechanical hypersensitivity in several pathological pain syndromes,
we wondered whether rescuing local astrocyte dysfunction in this
brain area could ameliorate the increased nocifensive responses
detected in FHM2 mice upon NTG treatment (23). To that end, we
expressed WT Atp1a2 (AAV.hGFAP.ATP1A2) in Cg astrocytes
of FHM2 mice. As a control, FHM2 mice were injected with AAV.
hGFAP.eGFP. Three weeks after viral injections, we triggered facial
mechanical sensitivity by NTG injections (5mg kg−1) and per-
formed facial von Frey tests as described above (Fig.5,FandG).
We found that rescuing the astrocytic loss-of-function FHM2
mutation locally in the Cg was sufficient to attenuate the acute
hypersensitive phenotype induced by NTG in FHM2 mice (Fig.5H
and fig. S9), suggesting that the local astrocyte dysfunction in
the Cg is implicated in orofacial pain sensitivity [AUC: control,
148.8±9.62 (N=14 mice) and rescue, 91.40±13.98 (N=13 mice;
**P=0.0021)].
Together, our results demonstrate that astrocytes in a genetic
migraine model display altered glutamate and K+ clearance in
the Cg, which facilitate neuronal NMDA spike generation and
synaptically evoked AP firing. We report that these alterations are
pathologically relevant since local rescue of the astrocytic dysfunc-
tions reduces facial hypersensitivity induced by a migraine-relevant
pain trigger.
Inj. Inj. **P = 0.001
AUC
Saline
NTG (10 mg kg−1)
Wild type FHM2 KI mice
A
B
NTG (10 mg kg−1)
Saline
AUC
AUC
Saline
NTG (5 mg kg−1)
Inj. Inj.
**P = 0.006
P = 0.118
***
***
***
*
D
C
Wild type
AUC
**P
= 0.002
***
E
F
Time (min)
0
50
100
150
200
250
BL1
BL2
30
60
120
180
0
2
4
6
Orofacial pain score
Time (min)
Orofacial pain score
BL1
BL2
30
60
120
180
0
2
4
6
***
*
AUC
Saline
NTG (10 mg kg−1)
NTG (10 mg kg−1)
NTG (5 mg kg−1)
NTG (5 mg kg−1)
0
50
100
150
200
250 **P = 0.002
Saline
0
50
100
150
200
250
0
50
100
150
200
250
Time (min)
BL1
BL230
60
120
180
0
2
4
6
Orofacial pain score
Time (min)
Orofacial pain score
BL1
BL2
30
60
120
180
0
2
4
6
NTG (5 mg kg−1)
AAV5/2.hGFAP.eGFP (control)
AAV5/2.hGFAP.ATP1A2 (rescue)
**
0
50
100
150
200
250
AAV5/2.hGFAP.eGFP (control)
or
AAV5/2.hGFAP.AT P1A2 (rescue)
FHM2 KI mice
21 d.p.i.
Wild-type or FHM2 KI mice
NTG (NO donor) or saline (i.p.)
Bilateral in cingulate cortex
Saline
NTG (10 mg kg−1)
Saline
NTG (5 mg kg−1)
Saline
FHM2 KI mice
Mechanical stimulation
AAV.eGFP
AAV.ATP1A
2
NTG (5 mg kg−1)
0.1-g filament
NTG (5 mg kg−1)
0.1-g filament
Mechanical stimulation
H
G
Time (min)
Orofacial pain score
BL1
BL2
30
60
120
180
0
2
4
6
0.1-g filament
Fig. 5. Local rescue of astrocyte dysfunction in the Cg reduces facial pain in FHM2
mice. (A) Schematic illustration of the experimental design. (B) Time course showing
the orofacial pain score in WT mice injected with either saline (gray) or NTG (black).
NTG (10 mg kg−1) elicits hypersensitivity to touch at 30, 60, and 120 min following in-
jection. (C) Same as (B) but for FHM2 mice injected with saline (gray) or NTG (red). NTG
(10 mg kg−1) evokes higher orofacial pain scores at 30, 60, and 120 min following injec-
tion. (D) A lower dose of NTG (5 mg kg−1) does not elicit a higher sensitivity in WT mice.
(E) In FHM2 mice, NTG (5 mg kg−1) evokes a higher orofacial pain score at 30 min after
injection. (F) Schematic illustration of the experimental design. (G) Three-dimensional
reconstruction of a cleared brain imaged with light-sheet microscopy showing the
volume and site of the injection in Cg after ex vivo fixation. Scale bar, 300 m. The bottom
left image shows a single coronal section, and dots indicate the center of the injection
volume of the viruses from six brains. (H) Time course showing the orofacial pain score
in FHM2 mice injected with the control virus (red) or the rescue virus (blue). NTG
(5 mg kg−1) only evoked a higher orofacial pain score at 30 min after injection in
contro l FHM2 mice and not in rescued FHM2. Data are means ± SEM. Two-way ANOVA
with Bonferroni post hoc test and two-tailed unpaired t test were used.
on June 5, 2020http://advances.sciencemag.org/Downloaded from
Romanos et al., Sci. Adv. 2020; 6 : eaaz1584 5 June 2020
SCIENCE ADVANCES | RESEARCH ARTICLE
8 of 13
DISCUSSION
Astrocytic dysfunction enhances NMDA-mediated dendritic
excitability in Cg of FHM2 mice
In this study, we first confirmed that in the adult Cg of FHM2 mice,
similar to the developing somatosensory cortex (3), 2 NKA dys-
function impairs both K+ and synaptically released glutamate up-
take by astrocytes. Western blot analysis performed in FHM2 mice
also revealed a significant reduction in the expression levels of 2
NKA and the GluT GLAST, which we previously reported to be
implicated in astrocyte-mediated glutamate uptake in this cortical
region (17). The reduced GLAST expression in FHM2 Cg could
suggest potential physical coupling between 2 NKA and GLAST in
Cg astrocytes, as was previously shown to occur between 2 NKA
and GLT-1in perisynaptic astrocytic processes of the somatosensory
cortex (9). As a consequence of this tight coupling, a reduced density
of GLT-1 transporters in perisynaptic astrocytic processes could be
previously detected with electron microscopy in the developing barrel
cortex of FHM2 mice (3). A local reduction of GLT-1 density spe-
cifically around synapses may remain undetected in Western blots
and be consistent with the unaltered expression of GLT-1in FHM2
Cg (28). With regard to the reported impairment of K+ clearance in
the Cg of FHM2 mice, this is likely to be directly ascribed to the re-
duced 2 NKA expression and function, which plays a major role in
K+ clearance following trains of high-frequency stimulation (29).
The slowdown in glutamate clearance by astrocytes prolonged
the presence of elevated glutamate levels in the extracellular space.
Increase in glutamate spillover enhances cortical excitatory neuro-
transmission, particularly NMDA receptor–mediated transmission
(17). Consistently, we found a facilitation of NMDA spike genera-
tion in tuft dendrites of L5 pyramidal cells both in FHM2 mice and
upon subsaturating concentrations of the GluT blocker dl-TBOA
(that reduces glutamate clearance to an extent similar to that pro-
duced by the FHM2 mutation) in WT mice. The probability of AP
firing following NMDA spikes was also increased in FHM2 mice. In
contrast, no changes were observed in spontaneous and evoked synaptic
transmission in FHM2 mice.
To establish a causal relationship between the astrocyte malfunc-
tion and the observed neuronal modifications in FHM2, we com-
pensated for the astrocyte dysfunction by expressing the WT form
of the Atp1a2 gene in the Cg of FHM2 mice. This intervention re-
versed the defective glutamate and K+ clearance by astrocytes.
Compensating the astrocyte dysfunction reduced the facilitation
of dendritic NMDA spike generation, thereby lowering the output
firing induced by these spikes. These findings demonstrate that NMDA
spike generation on tuft dendrites of L5 pyramidal cells and the sub-
sequent neuronal output are directly and dynamically affected by
astrocytic dysfunction.
NMDA spikes are believed to be the dominant mechanism by which
distal synaptic inputs lead to firing of pyramidal neurons in the cortex
(30). Since dendritic spikes increase the computational properties of
individual neurons (19,30), the facilitation of NMDA spike genera-
tion and the resulting firing of L5 pyramidal cells (the main output
cells of the Cg) could greatly influence network activity in down-
stream cortical and subcortical areas involved in pain processing.
The altered cellular functions in the Cg of FHM2 mice lead
to hypersensitivity to a migraine-relevant pain trigger
In migraineurs, NTG administration induces a delayed migraine-
like headache with associated features such as premonitory symptoms
and allodynia (24). NTG-induced hyperalgesia in animals provides
a behavioral model of the NTG-induced allodynia observed in
migraineurs during the attack (25). Using this model, we found that
relatively low doses of NTG trigger hypersensitivity to facial mechanical
stimulation in FHM2 mice, while they are ineffective in WT mice. A
similar finding was previously reported for another genetic mouse
model of migraine (26). Local rescue of the astrocytic defect by
expressing the WT Atp1a2 gene in the Cg of FHM2 mice strongly
reduced their increased nociceptive response upon NTG treatment.
This finding is consistent with the conclusion that the hypersensitivity
of FHM2 mice to a migraine-relevant trigger is largely due to altered
neural function in the Cg.
The Cg involvement in migraine pathophysiology
The anterior Cg (ACC) and midcingulate cortex (MCC) play a key
role in pain processing (23,31). The ACC is consistently activated
in humans and animal models upon nociceptive stimuli (32), and
neuronal plasticity in the ACC is correlated with the development
of chronic pain (21,23). The ACC is also among the regions that are
activated during spontaneous migraine attacks (33) and during the
premonitory phase of the delayed migraine-like headache induced
by NTG infusion in migraineurs (34). Moreover, functional imaging
studies show increased activation of both the ACC (12) and the MCC
(13,35) in response to noxious (including trigeminal) stimulation
in migraineurs during the interictal period. Migraineurs show in-
creased activation of the MCC that correlates with increased pain
rating (sensitization) during repeated trigeminal noxious stimula-
tion, while healthy controls show decreased MCC activation and
pain rating (habituation) (13). However, the underlying mechanism
of this hyperactivity and its potential involvement in cranial pain
induction was still elusive. We report an astrocyte-mediated facili-
tation of NMDA spike generation and a subsequent increase of L5
pyramidal cell firing in the Cg of FHM2 mice, which may be potentially
involved in pain generation and/or sensitization of the pain process-
ing system in familial migraine. The evidence that astrocyte dysfunc-
tion in the Cg can specifically increase facial tactile sensitivity to a
pain trigger supports the notion that the Cg is a critical hub in pain
processing and may additionally gate the activation of cranial pain
pathways. Whether the ACC, the MCC, or both mediate this remains
unclear. The ACC is broadly connected to the salience network and
projects to the periaqueductal gray, rostral ventromedial medulla,
and dorsal horn (5). An interesting pathway from the MCC to the
posterior insula (and involving descending serotoninergic facilitation
of nociception from the raphe magnus nucleus) has been shown to
be necessary and sufficient for the induction and maintenance of
pain sensitization and could also be involved in the observed behavior
(31). Therefore, heightened activity in the ACC and/or MCC could
have broad effects on migraine-relevant pain perception and descend-
ing modulatory circuits, which could lead to a loss of pain inhibition
and/or to pain facilitation. It would therefore be intriguing to test
how the rescue of the astrocytic mutation in the ACC or MCC affects
the activity of those downstream regions crucial for the development
or sensitization of cranial pain.
Our data support the idea that FHM2-associated astrocytic dys-
function in particular brain regions may engender different migraine-
relevant functional consequences. In the somatosensory cortex, it
lowers the CSD threshold with a potential impact on aura occurrence
and cranial nociceptor activation (3,10). CSD facilitation is a common
feature of all genetic models of migraine that have been investigated
on June 5, 2020http://advances.sciencemag.org/Downloaded from
Romanos et al., Sci. Adv. 2020; 6 : eaaz1584 5 June 2020
SCIENCE ADVANCES | RESEARCH ARTICLE
9 of 13
so far (26,36,37). In the hippocampus, the FHM2 mutation causes
abnormal region-dependent synaptic plasticity, which might underlie
some of the memory deficits observed in FHM2 patients (38). We
demonstrate instead that Cg astrocyte dysfunction in FHM2 mice
leads to hypersensitivity to a migraine-relevant trigger.
In conclusion, we provide evidence that astrocyte dysfunction in
the Cg, a nonsensory cortical area, is implicated in heightened
sensitivity to head pain triggers and may be involved in pain gener-
ation and/or sensitization of the pain processing system in familial
migraine. Understanding the cellular and molecular nature of
circuit-specific network dysfunctions associated with familial migraine
might be key to shed light on incompletely understood aspects of
migraine pathophysiology.
MATERIALS AND METHODS
Animals
Experiments were performed using adult (5 to 9 weeks old) hetero-
zygous KI mice harboring the W887R FHM2 mutation [Atp1a2+/R887
mice; (10)] and their WT littermates (background C57BL/6J; male
and female in equal or near-equal number). Adult mice were group-
housed up to five in filter-top cages with a standard 12-hour lig ht/ 12-h our
dark cycle and food and water available ad libitum. Permission for
animal experiments was obtained from the Tierversuchskommission
of the canton of Zurich, Zurich, Switzerland. All animal experiments
complied with the relevant ethical regulations.
Chemicals and drugs
Reagents for artificial cerebrospinal fluid (ACSF) and internal solution s,
biocytin, 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione
(NBQX), and picrotoxin were obtained from Sigma-Aldrich. 6-cyano-
7-nitroquinoxaline-2,3-dione (CNQX), AP-V, dl-TBOA, and d-serine
were obtained from Tocris. NBQX, CNQX, and dl-TBOA were dis-
solved in dimethyl sulfoxide. Tetrodotoxin (TTX) was obtained from
Abcam. NTG was obtained from Sigma-Aldrich. Picrotoxin was disso lved
in ethanol (EtOH). AP-V, d-serine, and TTX were dissolved in ddH2O.
Acute brain slice preparation
Mice were briefly anesthetized with isoflurane and decapitated. The
brain was quickly removed and transferred to an ice-cold solution
containing 65 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM
NaHCO3, 7 mM MgCl2, 0.5 mM CaCl2, 25 mM glucose, and 105 mM
sucrose saturated with 95% O2 and 5% CO2; coronal slices (350 m thick)
containing the ACC were cut from the tissue block with a vibratome
(HM 650, Microm). Slices were then transferred to a recovery solu-
tion containing 130 mM K-gluconate, 15 mM KCl, 0.2 mM EGTA,
20 mM Hepes, and 25 mM glucose for 2min before being kept in
oxygenated ACSF (315 mosm) saturated with 95% O2 and 5% CO2
and containing 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 25 mM
NaHCO3, 1 mM MgCl2, 2 mM CaCl2, and 25 mM glucose at 34°C
for 25min and then at room temperature until use. Slices used for
two-photon glutamate imaging and astrocytic patch-clamp recordings
were loaded with SR-101 (1M) for 15min at 34°C before being
kept in ACSF at room temperature.
Electrophysiological recordings
Whole-cell recordings from astrocytes
Recordings were performed as previously described in (17). Individual
slices were transferred to a recording chamber perfused with oxygenated
ACSF, at a flow rate of 1 to 2 ml/min at 32° to 34°C. Whole-cell re-
cordings were taken from L1 astrocytes in the Cg. Unless otherwise
stated, cell bodies of astrocytes were visualized using an astrocyte-
specific dye (SR-101; see above) that was excited with wLS broad-
band light-emitting diode illumination (460 nm), and images were
acquired with Retiga R1 camera using Ocular software (QImaging,
Germany) with a 40× water immersion objective. In addition, astro-
cytes were recognized by their hyperpolarized resting membrane
potential, their linear current-voltage relationship, their inability to
generate APs, and their low input resistance. Recordings were taken
with borosilicate glass pipettes (4 to 8megohm) containing the fol-
lowing internal solution: 115 mM K-gluconate, 6 mM KCl, 5 mM
glucose, 7.8 mM Na-phosphocreatine, 4 mM Mg-ATP (adenosine
triphosphate), 0.4 mM Na-GTP (guanosine triphosphate) [pH 7.25
with KOH; osmolarity, 295 mosm (readjusted with sucrose when
necessary)]. Recordings were performed using MultiClamp 700B
amplifier, and data were acquired with a Digidata 1550A 16-bit
board (all from Molecular Devices). For the recording of STCs, the
extracellular solution contained antagonists of NMDA receptors
(AP-V; 50 M), AMPA receptors (CNQX or NBQX; 10 M), and
-aminobutyric acid type A (GABAA) receptors (picrotoxin; 100 M).
Astrocytes were held at −80 mV, and STCs were evoked by single-
pulse stimulation or by trains of 10 and 11 pulses at high frequen-
cies (50and then 100 Hz) every 20 s. The K+ inward current was
characterized as the slowly decaying inward current elicited in as-
trocytes upon high-frequency synaptic stimulations. Every protocol
was repeated at least five times and then averaged and analyzed.
Currents were evoked by focal electrical stimulation (bipolar, 100 s,
8.5 V) through a theta glass pipette placed in L1, in the proximity of the
recorded astrocyte. Access resistance was monitored (<16megohm),
and recordings with an access resistance changing more than 30%
between the beginning and the end of the recording were discarded.
Resting membrane potential and input resistance were monitored
for analysis of electrophysiological properties of astrocytes. The de-
cay kinetics of the last pulse of the trains were analyzed by subtract-
ing the current elicited by 10 pulses from that elicited by the 11th
pulse.
Whole-cell recordings from L5 pyramidal neurons
Somas were patched with borosilicate glass pipettes (2.2 to 4megohm).
Cells were clamped at −70 mV, and focal synaptic stimulation was
performed through a theta patch pipette located close to the selected
apical tuft dendritic segments in L1. NMDA spikes were evoked by
applying paired-pulse stimulations of 50Hz of increasing stimula-
tion intensities (from 1 to 2.5 mA or 5.5 mA). Recordings with an
access resistance >15 megohm were discarded. The following internal
solution was used: 130 mM K-gluconate, 5 mM KCl, 10 mM Hepes,
10 mM phosphocreatine, 4 mM Mg-ATP, 0.3 mM GTP, and biocytin
(1.5 mg/ml) (pH 7.3 with KOH; osmolarity, 294 mosm). To visualize the
dendrites, patch pipettes also contained Alexa Fluor 488. Recordings
were performed in the presence of GABAA receptor blocker (picro-
toxin; 100 M) and d-serine (10 M).
For mEPSCs, somata were targeted for recording with borosilicate
glass pipettes (2.2 to 4megohm) containing the following: 130 mM
gluconic acid, 130 mM CsOH, 5 mM CsCl, 10 mM Hepes, 1.1 mM EGTA,
10 mM Na-phosphocreatine, 4 mM Mg-ATP, and 0.3 mM Na-GTP.
The pH of the intracellular solution was adjusted to 7.3 with CsOH,
and biocytin (1.5mg ml−1) was added for the reconstruction of neurons.
Cells were held at −70 mV, and synaptic responses were recorded in
the presence of picrotoxin (100 M) and TTX (1 mM). Recordings
on June 5, 2020http://advances.sciencemag.org/Downloaded from
Romanos et al., Sci. Adv. 2020; 6 : eaaz1584 5 June 2020
SCIENCE ADVANCES | RESEARCH ARTICLE
10 of 13
with an unstable baseline or a holding current less than −400 pA
were rejected. Currents were filtered off-line using a Butterworth
low-pass filter (2 kHz) and analyzed in 1-min epochs using the Mini
Analysis Program 6.0.7 (Synaptosoft Inc., USA). Recordings with
leak current increasing more than 100 pA and access resistance
changing more than 30% between the beginning and the end of the
recording were discarded. At least 100 events were analyzed for every
condition. Events were identified as mEPSCs by setting the event
detection threshold at least twice the baseline noise level and by
checking that events had (i) rise times faster than the decay time, (ii) rise
times greater than 0.5 ms, and (iii) decay times greater than 1.5 ms.
Events not fitting the above parameters were rejected. Event amplitudes,
interevent intervals, and rise and decay times were first averaged
within each experiment and regrouped by condition. The resulting
means were averaged between experiments. Single-cell properties
(access resistance, membrane capacitance, etc.) were analyzed with
Clampfit 10.5 (Axon Instruments, Union City, CA). Graphs were
made using GraphPad Prism and Illustrator 15.1.0 (Adobe). For
evoked AMPA-mediated excitatory postsynaptic currents (eEPSCs),
cells were held at −70 mV and focal synaptic stimulation was
performed through a theta patch pipette located close to the tuft
dendrites in L1. AMPA eEPSCs were evoked by applying either a
single stimulation or paired-pulse stimulations at 20Hz. Recordings
were acquired in the presence of picrotoxin (100 M) and AP-V (50 M)
in ACSF.
Biocytin labeling
For some experiments, internal solutions used for recording of py-
ramidal cells contained biocytin (1.5 mg/ml) that diffused in the
cells for at least 10 min, as previously described in (17). Briefly, slices
(350 m) containing the recorded cell were then fixed in 4% para-
formaldehyde (PFA) at +4°C overnight. The following day, slices were
washed in phosphate-buffered saline (PBS) and 274 mM NaCl be-
fore being transferred to a blocking solution containing 10% nor-
mal goat serum in 0.3% Triton-PBS and 274 mM NaCl for 1 hour.
Afterward, slices were put in a blocking solution containing Alexa
Fluor 647–conjugated streptavidin (1:700; Jackson ImmunoResearch
Europe Ltd.; code: 016-600-084) for 2 hours. Slices were then
washed in PBS and 274 mM NaCl before being mounted on Superfrost
Plus slides. Images were acquired on a Zeiss LSM 710 Pascal confocal
microscope using a 0.9 numerical aperture ×10 Plan-Apochromat
objective (for L5 pyramidal cells) and the ZEN 2012 software (Carl Zei ss).
Whenever applicable, contrast and illumination were adjusted in
ImageJ. Presented images are Z projections.
AAV5/2 generation and injections in vivo
The mAtp1a2 gene (WT form of 2 NKA for compensation exper-
iments) and the EGFP gene (for control experiments) were cloned
into plasmid backbones containing a shorted glial fibrillary acidic
protein (HgfaABC1D) promoter as in (22). These plasmids were
packaged into AAV serotype 5 [AAV-5/2-hGFAP-mATP1A2-bGH p(A)
and AAV5/2- hGFAP-hHBb1/E-EGFP-bGHp(A)] by the Viral Vector
Facility of the University of Zurich. All electronic information con-
cerning the plasmids and viruses is available online in the viral vector
repository (https://vvf.ethz.ch). FHM2 KI mice were used in all
experiments in accordance with institutional guidelines.
All surgical procedures were conducted under general anesthesia
using continuous isoflurane inhalation (induction at 5% and main-
tenance at 1 to 3%). Following induction of anesthesia, the mice
were placed into a motorized stereotaxic frame (David Kopf Instru-
ments and NeuroStar) using adjustable ear bars and an anesthesia
mask with an incisor bar. The animal was maintained at a physio-
logical temperature using a heating mat placed between the animal
and the frame. Vitamin C ointment was applied to the eyes to prevent
corneal drying during the operation. Mice were subcutaneously
administered buprenorphine (0.2 mg/kg) before surgery. The fur
between the ears was shaved, scrubbed with EtOH and betadine,
and dried, and a longitudinal incision of approximately 3 to 5mm
was made in the skin above the skull. For unilateral injections, a single
hole was drilled through the skull directly above the Cg on one
hemisphere (stereotaxic coordinates with respect to bregma: 0.75mm
anterior, 0.3mm lateral, and 1.25mm ventral). For control experiments,
0.5 l of the control virus AAV5/2- hGFAP-hHBb1/E-EGFP-bGHp(A)
was injected through a glass pipette. Since AAV vectors size is limited
to ~4.7 kb, it was not possible to include a reporter gene in the rescue
virus AAV-5/2-hGFAP-mATP1A2-bGHp(A). Therefore, to visualize
the injection site, we injected a mixture of 0.5 l of the two viruses
with a ratio of 2:1 of the rescue virus to the control virus containing
eGFP. Glass pipettes were left in place for at least 10min follow-
ing infusion of the virus. Surgical wounds were closed with single
5-0 nylon sutures. Following surgery, animals were closely monitored.
Mice were euthanized 15 to 25 days after surgery for electrophysiology
experiments or were used for behavioral experiments. When assess-
ing behavior, AAVs were injected bilaterally. The experimenter was
blinded to which AAV was injected.
Two-photon glutamate imaging
Mice aged between 4 and 6 weeks were injected with 0.3 to 0.5 l of
AAV2/1.GFAP.iGluSnFr.WPRE.SV40 (Penn Vector; provided by
L. Looger, Janelia Farm) unilaterally into the Cg through a glass
pipette as described above. Fifteen to 21 days following the virus
injections, coronal brain slices (350 m) containing the Cg were
obtained as previously described (21). Imaging was performed as
described in (17). Briefly, a galvanometer-based two-photon laser
scanning system was used to image extracellular glutamate (16×
objective; zoom, 6; excitation wavelength, 900 nm; 64pixels by 64 pixels
per image; acquisition rate, 19.2 Hz). Astrocytes were visualized using
SR-101. Synaptic glutamate release was elicited by trains of 10 pulses
at high frequency (50 or 100 Hz) every 20 s delivered via a theta
glass pipette (bipolar, 100 s; stimulation intensity, 3 to 5 V) placed
in the inner L1. To visualize the theta glass pipette, it was filled with
ACSF containing 1 M SR-101. Note that SR-101 staining does not
affect the kinetics of iGluSnFr transients nor of STCs as shown in
(17). Moreover, when we doubled the acquisition rate to 38.4 Hz,
the iGluSnFr decay kinetics remained unaltered, indicating that the
image acquisition rate used was sufficient to detect the monitored
changes. All solutions contained 10M NBQX or CNQX, 50M
AP-V, and 100M picrotoxin, and temperature was kept between
32° and 34°C during imaging. Ten consecutive sweeps were acquired
and subsequently analyzed using ImageJ. Fluorescence emission
was collected from an ROI (diameter, 34 m) 10 to 40 m away
from the stimulation pipette. The average background value was
derived from a region within the field of view that was free
of clearly visible iGluSnFr (typically in the contralateral hemi-
sphere) and subtracted from the fluorescence intensity of the ROIs
for each frame. Traces were then averaged, and decay tau was cal-
culated by fitting a single exponential function using Igor Pro
(WaveMetrics).
on June 5, 2020http://advances.sciencemag.org/Downloaded from
Romanos et al., Sci. Adv. 2020; 6 : eaaz1584 5 June 2020
SCIENCE ADVANCES | RESEARCH ARTICLE
11 of 13
Behavioral analysis
Orofacial von Frey
Behavioral testing was performed in a dimly lit and quiet room by
the same female experimenter. Throughout the week before the first
behavioral testing, mice were habituated to handling by the experi-
menter. Each mouse was placed in Plexiglas cages of about 20cm by
20cm sitting on a metal grid ground floor. Mice were allowed to
accommodate to the cage for 1 hour before the testing. In all experime nts,
0.025- and 0.01-g von Frey filaments were used. The first tests were
performed using the lower-force filament. Filaments were applied
to the orofacial area close to the whisker pad or close to a 90° angle
until bent, in three series of four pokes, in the middle or on either
left or right side of the snout. After a total of 12 pokes with the
0.025-g filament, the stimulation with the 0.01-g filament was
applied in the same way. The responses recorded were as follows:
unilateral or bilateral forepaw swipes across the face (1 point each),
continuous forepaw swipes (three or more: 1.5 points), aggression/
biting of the probe following stimulus (0.25 points), and clear with-
drawal of the head from the stimulus (0.25 points) as described
in (27). Before the injection of NTG (5 or 10 mg/kg) or 0.9% saline
(intraperitoneally), baseline 1 was recorded several hours or a day
before the experiment and baseline 2 was immediately before intra-
peritoneal injections. Testing was carried out at 30, 60, 120, and
180min after injection. The experimenter was blinded to the treat-
ment in all behavioral experiments.
Locomotor activity
NTG (5 mg/kg, i.p.) or saline was administered 30min before testing.
Locomotor activity was measured in an open-field arena (radius,
10 cm) equipped with four pairs of light beams and photosensors
and analyzed for the time interval between 10 and 60min after NTG
or saline administration.
Brain clearing and light-sheet microscopy
After fixation and dehydration
Mice were anesthetized with isoflurane and decapitated. Entire brains
were extracted, briefly washed in PBS to remove excessive blood, and
postfixed in 4% PFA for 2 days at 4°C. After fixation, brains were washed
two times in PBS and subsequently dehydrated in increasing alcohol
concentrations [30, 50, 70, 80, 90, 96, and 100% EtOH (each adjusted
to pH 9.5)] for a day each (39). Tissue shrinkage of up to 50% was
observed during the dehydration process. After dehydration, brains were
transferred into a clearing solution of benzyl alcohol and benzyl benzoate
(1:2) solution (BABB) in separate glass vials on a gentle shaking or rotat-
ing cycle under a chemical hood for a minimum of 1 day until transparen t.
Imaging
Cleared brains were transferred to an immersion cuvette containing
BABB and placed in the imaging reservoir of the microscope. Images
were acquired using the mesoscale single-plane illumination micro-
scope mesoSPIM system (www.mesospim.org) with an Olympus MVX10
macroscope in the detection path and an MVPLAPO 1× objective
(40). The fluorescent signal in the sample was recorded at ×1.6 magnifica-
tion by moving the sample through the light sheet in 4-m steps. The
sample was illuminated from one side using Toptica multi-laser engine
laser at excitation wavelengths of 488 and 640nm. Postprocessing of
images was carried out using ImageJ and Imaris software (Bitplane).
Western blots
Two weeks following stereotaxic viral injections in the Cg, tissue
of the Cg was rapidly dissected and immediately frozen on dry ice
and stored at −80°C until used. On the day of the experiment, the
tissue (10 to 20 mg) was thawed and homogenized by sonication in
20 volumes of 20 mM tris (pH 7.4) containing the protease inhibitor
cocktail cOmplete Mini (Roche Diagnostics). The homogenate was
centrifuged for 10min at 1000g, and the supernatant was recovered.
After total protein quantification using the Bradford protein assay
(Bio-Rad), the samples were incubated with Laemmli sample buffer
(Bio-Rad) for 30min at 37°C. Aliquots containing 10g (GLAST)
or 15 g of protein (ATPase 2 and GLT-1) were subjected to SDS–
polyacrylamide gel electrophoresis using 10% (GLAST and GLT-1)
or 7.5% (ATPase 2) mini gels (Mini-PROTEAN 3, Bio-Rad). Pro-
teins were transferred onto nitrocellulose membranes in a Trans-
Blot semi-dry transfer cell (Bio-Rad) at 15 V for 60min using 39 mM
glycine, 48 mM tris, 1.3 mM SDS, and 20% methanol as transfer
buffer. After blotting, the transferred proteins were stained with
REVERT Total Protein Stain (LI-COR) and immediately imaged
using the Odyssey CLx imager (LI-COR). For immunodetection,
the blots were blocked for 1 hour in PBS containing 5% nonfat dry
milk at room temperature, followed by incubation at 4°C overnight
with anti–2 NKA antibodies (1:1000; rabbit polyclonal, Sigma-
Aldrich, catalog no. 07-674), anti–GLT-1 antibodies (1:1000; rabbit
polyclonal, knockout verified, Synaptic Systems, catalog no.
250203), or anti-GLAST antibodies (1:10,000; rabbit polyclonal,
knockout verified, Synaptic Systems, catalog no. 250113), diluted in
PBST [PBS (pH 7.4) and 0.05% Tween 20] containing 5% nonfat
dry milk. The blots were then washed five times for 5 min with Tris
Buffered Saline + Tween 20 (TBST) (10 mM Tris, pH 7.4, 150 mM
NaCl, 0.05% Tween 20) and incubated with secondary antibodies
(1:20,000; goat anti-rabbit Alexa Fluor Plus 800) for 1 hour at room
temperature. Following extensive washing (see above), immuno-
reactivity was detected using the Odyssey CLx imager (LI-COR).
Immunoreactivity was quantified with the Image Studio software
(LI-COR) and normalized to total protein in the corresponding
lanes. Each antibody was tested for its linear detection range using
different protein and antibody concentrations.
Immunohistochemistry and image analysis
Three weeks after stereotaxic viral injections in the Cg, FHM2 KI mice
were anaesthetized with pentobarbital (160mg kg−1, i.p.) before
transcardiac perfusion with 20ml of phosphate buffer followed by
100ml of 4% ice-cold PFA [in 0.1M sodium phosphate buffer (pH 7.4)].
Brain tissue was postfixed for 4 hours with 4% PFA on ice and cryo-
protected in 30% sucrose solution (in 0.1M sodium phosphate buffer)
overnight at 4°C. Brains were embedded in NEG50 frozen section
medium (Richard-Allan Scientific) and cut into 40-m free-floating
sections (Hyrax KS 34 microtome, Carl Zeiss). Brain sections were
left in antifreezing solution at −20°C until use. Following incubation
in blocking solution (PBS, 0.3% Triton X-100, and 10% normal
donkey serum) for 1 hour, brain sections were incubated at 4°C
overnight in a primary antibody solution (PBS, 0.3% Triton X-100,
and 10% normal donkey serum) containing combinations of the
following antibodies: chicken anti-GFP (1:1000; LifeTech,
AB_2534023), guinea pig anti-NeuN (1:1000; Synaptic Systems,
AB_2619988), and rabbit anti-S100B (1:700; Abcam, AB_52642).
Three washing steps of 10min each in PBS were performed before
incubating brain sections with secondary antibodies (1:500): Cyanine
Cy3 donkey anti-rabbit (Jackson ImmunoResearch, AB_2307443),
Alexa Fluor 647 donkey anti-guinea pig (Jackson ImmunoResearch,
AB_2340477), and Alexa Fluor 488 donkey anti-chicken (Jackson
on June 5, 2020http://advances.sciencemag.org/Downloaded from
Romanos et al., Sci. Adv. 2020; 6 : eaaz1584 5 June 2020
SCIENCE ADVANCES | RESEARCH ARTICLE
12 of 13
ImmunoResearch, AB_2340376) for 90min at room temperature in
PBS supplemented with 0.3% Triton X-100. Images were taken with an
LSM 800 with Airyscan confocal microscopes (Carl Zeiss) controlled
with ZEN 2.3 (blue edition) software and using a Plan-Apochromat
×40/1.4 Oil DIC M27 oil-immersion objective. Z stack images of
10 optical sections and 1.5-m step size were used for the analysis of
fluorescence colocalization and to create maximum intensity pro-
jections images. Images were processed using ImageJ software.
Quantification and statistical analysis
Data are displayed as means±SEM in all experiments except for the
Western blot data that are displayed as means±SD. Statistical details
can be found in Results, figures, and figure legends. Statistical com-
parisons were made with two-tailed paired or unpaired t tests, one-
way analysis of variance (ANOVA) with Bonferroni post hoc test,
one-way ANOVA with Tukey post hoc test, or two-way repeated-
measures ANOVA test. All graphs and statistical tests were performed
using GraphPad Prism, and figures were prepared using Adobe
Illustrator CS5. P values less than 0.05 were considered statistically
significant.
SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/
content/full/6/23/eaaz1584/DC1
View/request a protocol for this paper from Bio-protocol.
REFERENCES AND NOTES
1. M. Santello, N. Toni, A. Volterra, Astrocyte function from information processing
to cognition and cognitive impairment. Nat. Neurosci. 22, 154–166 (2019).
2. X. Tong, Y. Ao, G. C. Faas, S. E. Nwaobi, J. Xu, M. D. Haustein, M. A. Anderson, I. Mody,
M. L. Olsen, M. V. Sofroniew, B. S. Khakh, Astrocyte Kir4.1 ion channel deficits contribute
to neuronal dysfunction in Huntington’s disease model mice. Nat. Neurosci. 17, 694–703
(2014).
3. C. Capuani, M. Melone, A. Tottene, L. Bragina, G. Crivellaro, M. Santello, G. Casari, F. Conti,
D. Pietrobon, Defective glutamate and K+ clearance by cortical astrocytes in familial
hemiplegic migraine type 2. EMBO Mol. Med. 8, 967–986 (2016).
4. D. Pietrobon, M. A. Moskowitz, Pathophysiology of migraine. Annu. Rev. Physiol. 75,
365–391 (2013).
5. K. C. Brennan, D. Pietrobon, A systems neuroscience approach to migraine. Neuron 97,
1004–1021 (2018).
6. T. J. Schwedt, C. C. Chiang, C. D. Chong, D. W. Dodick, Functional MRI of migraine.
Lancet. Neurology 14, 81–91 (2015).
7. A. Russo, A. Tessitore, F. Esposito, L. Marcuccio, A. Giordano, R. Conforti, A. Truini,
A. Paccone, F. d’Onofrio, G. Tedeschi, Pain processing in patients with migraine:
An event- related fMRI study during trigeminal nociceptive stimulation. J. Neurol. 259,
1903–1912 (2012).
8. M. De Fusco, R. Marconi, L. Silvestri, L. Atorino, L. Rampoldi, L. Morgante, A. Ballabio,
P. Aridon, G. Casari, Haploinsufficiency of ATP1A2 encoding the Na+/K+ pump 2
subunit associated with familial hemiplegic migraine type 2. Nat. Genet. 33,
192–196 (2003).
9. M. Melone, C. Ciriachi, D. Pietrobon, F. Conti, Heterogeneity of astrocytic and neuronal
GLT-1 at cortical excitatory synapses, as revealed by its colocalization with Na+/K+-ATPase
Isoforms. Cereb. Cortex 29, 3331–3350 (2019).
10. L. Leo, L. Gherardini, V. Barone, M. De Fusco, D. Pietrobon, T. Pizzorusso, G. Casari,
Increased susceptibility to cortical spreading depression in the mouse model of familial
hemiplegic migraine type 2. PLOS Genet. 7, e1002129 (2011).
11. I. Ellerbrock, A. K. Engel, A. May, Microstructural and network abnormalities in headache.
Curr. Opin. Neurol. 26, 353–359 (2013).
12. A. Russo, F. Esposito, F. Conte, M. Fratello, G. Caiazzo, L. Marcuccio, A. Giordano,
G. Tedeschi, A. Tessitore, Functional interictal changes of pain processing in migraine
with ictal cutaneous allodynia. Cephalalgia 37, 305–314 (2016).
13. A. Stankewitz, E. Schulz, A. May, Neuronal correlates of impaired habituation in response
to repeated trigemino-nociceptive but not to olfactory input in migraineurs: An fMRI
study. Cephalalgia 33, 256–265 (2012).
14. N. Cholet, L. Pellerin, P. J. Magistretti, E. Hamel, Similar perisynaptic glial localization
for the Na+, K+-ATPase 2 subunit and the glutamate transporters GLAST and GLT-1
in the rat somatosensory cortex. Cereb. Cortex 12, 515–525 (2002).
15. J. S. Marvin, B. G. Borghuis, L. Tian, J. Cichon, M. T. Harnett, J. Akerboom, A. Gordus,
S. L. Renninger, T. W. Chen, C. I. Bargmann, M. B. Orger, E. R. Schreiter, J. B. Demb,
W. B. Gan, S. A. Hires, L. L. Looger, An optimized fluorescent probe for visualizing
glutamate neurotransmission. Nat. Methods 10, 162–170 (2013).
16. M. P. Parsons, M. P. Vanni, C. L. Woodard, R. Kang, T. H. Murphy, L. A. Raymond, Real-time
imaging of glutamate clearance reveals normal striatal uptake in Huntington disease
mouse models. Nat. Commun. 7, 11251 (2016).
17. J. Romanos, D. Benke, A. S. Saab, H. U. Zeilhofer, M. Santello, Differences in glutamate
uptake between cortical regions impact neuronal NMDA receptor activation. Commun. Biol.
2, 127 (2019).
18. J. R. Chalifoux, A. G. Carter, Glutamate spillover promotes the generation of NMDA spikes.
J. Neurosci. 31, 16435–16446 (2011).
19. L. M. Palmer, A. S. Shai, J. E. Reeve, H. L. Anderson, O. Paulsen, M. E. Larkum, NMDA spikes
enhance action potential generation during sensory input. Nat. Neurosci. 17, 383–390
(2014).
20. J. Schiller, G. Major, H. J. Koester, Y. Schiller, NMDA spikes in basal dendrites of cortical
pyramidal neurons. Nature 404, 285–289 (2000).
21. M. Santello, T. Nevian, Dysfunction of cortical dendritic integration in neuropathic pain
reversed by serotoninergic neuromodulation. Neuron 86, 233–246 (2015).
22. J. L. Stobart, K. D. Ferrari, M. J. P. Barrett, M. J. Stobart, Z. J. Looser, A. S. Saab, B. Weber,
Long-term in vivo calcium imaging of astrocytes reveals distinct cellular compartment
responses to sensory stimulation. Cereb. Cortex 28, 184–198 (2018).
23. T. V. P. Bliss, G. L. Collingridge, B.-K. Kaang, M. Zhuo, Synaptic plasticity in the anterior
cingulate cortex in acute and chronic pain. Nat. Rev. Neurosci. 17, 485–496 (2016).
24. I. Christiansen, L. L. Thomsen, D. Daugaard, V. Ulrich, J. Olesen, Glyceryl trinitrate induces
attacks of migraine without aura in sufferers of migraine with aura. Cephalalgia 19,
660–667 (1999).
25. E. A. Bates, T. Nikai, K. C. Brennan, Y. H. Fu, A. C. Charles, A. I. Basbaum, L. J. Ptácek,
A. H. Ahn, Sumatriptan alleviates nitroglycerin-induced mechanical and thermal
allodynia in mice. Cephalalgia 30, 170 (2009).
26. K. C. Brennan, E. A. Bates, R. E. Shapiro, J. Zyuzin, W. C. Hallows, Y. Huang, H.-Y. Lee,
C. R. Jones, Y.-H. Fu, A. C. Charles, L. J. Ptáček, Casein kinase i mutations in familial
migraine and advanced sleep phase. Sci. Transl. Med. 5, 183ra56 (2013).
27. A. Krzyzanowska, S. Pittolo, M. Cabrerizo, J. Sánchez-López, S. Krishnasamy, C. Venero,
C. Avendaño, Assessing nociceptive sensitivity in mouse models of inflammatory
and neuropathic trigeminal pain. J. Neurosci. Methods 201, 46–54 (2011).
28. U. Pannasch, D. Freche, G. Dallérac, G. Ghézali, C. Escartin, P. Ezan, M. Cohen-Salmon,
K. Benchenane, V. Abudara, A. Dufour, J. H. R. Lübke, N. Déglon, G. Knott, D. Holcman,
N. Rouach, Connexin 30 sets synaptic strength by controlling astroglial synapse invasion.
Nat. Neurosci. 17, 549–558 (2014).
29. R. D'Ambrosio, D. S. Gordon, H. R. Winn, Differential role of KIR channel and Na+/K+-pump
in the regulation of extracellular K+ in rat hippocampus. J. Neurophysiol. 87, 87–102
(2002).
30. G. Major, M. E. Larkum, J. Schiller, Active properties of neocortical pyramidal neuron
dendrites. Annu. Rev. Neurosci. 36, 1–24 (2013).
31. L. L. Tan, P. Pelzer, C. Heinl, W. Tang, V. Gangadharan, H. Flor, R. Sprengel, T. Kuner,
R. Kuner, A pathway from midcingulate cortex to posterior insula gates nociceptive
hypersensitivity. Nat. Neurosci. 20, 1591–1601 (2017).
32. T. D. Wager, L. Y. Atlas, M. A. Lindquist, M. Roy, C. W. Woo, E. Kross, An fMRI-based
neurologic signature of physical pain. N. Engl. J. Med. 368, 1388–1397 (2013).
33. S. K. Afridi, N. J. Giffin, H. Kaube, K. J. Friston, N. S. Ward, R. S. J. Frackowiak, P. J. Goadsby,
A positron emission tomographic study in spontaneous migraine. Arch. Neurol. 62,
1270–1275 (2005).
34. F. H. Maniyar, T. Sprenger, T. Monteith, C. Schankin, P. J. Goadsby, Brain activations
in the premonitory phase of nitroglycerin-triggered migraine attacks. Brain 137, 232–241
(2014).
35. T. J. Schwedt, C. D. Chong, C. C. Chiang, L. Baxter, B. L. Schlaggar, D. W. Dodick, Enhanced
pain-induced activity of pain-processing regions in a case-control study of episodic
migraine. Cephalalgia 34, 947–958 (2014).
36. A. Tottene, R. Conti, A. Fabbro, D. Vecchia, M. Shapovalova, M. Santello,
A. M. J. M. van den Maagdenberg, M. D. Ferrari, D. Pietrobon, Enhanced excitatory
transmission at cortical synapses as the basis for facilitated spreading depression
in Cav2.1 knockin migraine mice. Neuron 61, 762–773 (2009).
37. A. M. J. M. van den Maagdenberg, T. Pizzorusso, S. Kaja, N. Terpolilli, M. Shapovalova,
F. E. Hoebeek, C. F. Barrett, L. Gherardini, R. C. G. van de Ven, B. Todorov, L. A. M. Broos,
A. Tottene, Z. Gao, M. Fodor, C. I. de Zeeuw, R. R. Frants, N. Plesnila, J. J. Plomp,
D. Pietrobon, M. D. Ferrari, High cortical spreading depression susceptibility
and migraine-associated symptoms in Cav2.1 S218L mice. Ann. Neurol. 67, 85–98
(2010).
on June 5, 2020http://advances.sciencemag.org/Downloaded from
Romanos et al., Sci. Adv. 2020; 6 : eaaz1584 5 June 2020
SCIENCE ADVANCES | RESEARCH ARTICLE
13 of 13
38. A. de Iure, P. Mazzocchetti, G. Bastioli, B. Picconi, C. Costa, I. Marchionni, G. Casari,
A. Tozzi, D. Pietrobon, P. Calabresi, Differential effect of FHM2 mutation on synaptic
plasticity in distinct hippocampal regions. Cephalalgia 39, 1333–1338 (2019).
39. H. U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C. P. Mauch, K. Deininger, J. M. Deussing,
M. Eder, W. Zieglgänsberger, K. Becker, Ultramicroscopy: Three-dimensional visualization
of neuronal networks in the whole mouse brain. Nat. Methods 4, 331–336 (2007).
40. F. F. Voigt, D. Kirschenbaum, E. Platonova, S. Pagès, R. A. A. Campbell, R. Kastli,
M. Schaettin, L. Egolf, A. van der Bourg, P. Bethge, K. Haenraets, N. Frézel, T. Topilko,
P. Perin, D. Hillier, S. Hildebrand, A. Schueth, A. Roebroeck, B. Roska, E. T. Stoeckli,
R. Pizzala, N. Renier, H. U. Zeilhofer, T. Karayannis, U. Ziegler, L. Batti, A. Holtmaat,
C. Lüscher, A. Aguzzi, F. Helmchen, The mesoSPIM initiative: Open-source light-sheet
microscopes for imaging cleared tissue. Nat. Methods 16, 1105–1108 (2019).
Acknowledgments: We thank G. Casari for generating and providing the FHM2 KI mouse
model; J. C. Paterna and M. Rauch from the local Viral Vector Facility for help with the
production of the viruses; H. Johannssen for assembling the two-photon microscope and
helping with two-photon imaging; E. Platonova and the Center of Microscopy and Image
Analysis at UZH for assistance with light-sheet microscopy; T. Grampp for help with Western
blots; G. Albisetti and R. Ganley for advice on immunohistochemistry and proofreading the
manuscript; S. Gudmundsdottir for some electrophysiological experiments; W. B. Gan and
J. Cichon for providing the GFAP-iGluSnFr AAV viruses and training with cortical glutamate
imaging; and members of H.U.Z. group, F. Brandalise, R. Min, P. Bezzi, and B. Weber for
discussion. Funding: This work was supported by the Swiss National Science Foundation
grant PP00P3_176838, Novartis Foundation for medical-biological Research grant no. 17C157,
and the Hartmann-Müller Stiftung no. 2253 to M.S. and a Telethon grant GGP14234 and PRIN
2017ANP5L8 to D.P. Author contributions: J.R. and M.S. conceived the project, designed
the study, wrote the manuscript, performed two-photon imaging experiments, and analyzed
the data. J.R. performed patch-clamp experiments, behavioral experiments, light-sheet
imaging, and immunohistochemistry experiments; contributed to virus production; and
analyzed the data. D.B. performed and analyzed Western blot experiments. H.U.Z. contributed
to reagents and equipment and discussed the interpretation of the data. D.P. provided the
FHM2 KI mouse model, discussed the interpretation of the data, and contributed to the study
design. All authors edited and approved the final manuscript. M.S. supervised the whole
project. Competing interests: The authors declare that they have no competing interests.
Data and materials availability: All data needed to evaluate the conclusions in the paper are
present in the paper and/or the Supplementary Materials. Data, associated protocols, and
additional information regarding this paper are available to the reader upon request from the
authors and are located in the internal server of the Institute of Pharmacology and Toxicology
at the University of Zurich.
Submitted 16 August 2019
Accepted 25 March 2020
Published 5 June 2020
10.1126/sciadv.aaz1584
Citation: J. Romanos, D. Benke, D. Pietrobon, H. U. Zeilhofer, M. Santello, Astrocyte dysfunction
increases cortical dendritic excitability and promotes cranial pain in familial migraine. Sci. Adv.
6, eaaz1584 (2020).
on June 5, 2020http://advances.sciencemag.org/Downloaded from
familial migraine
Astrocyte dysfunction increases cortical dendritic excitability and promotes cranial pain in
Jennifer Romanos, Dietmar Benke, Daniela Pietrobon, Hanns Ulrich Zeilhofer and Mirko Santello
DOI: 10.1126/sciadv.aaz1584
(23), eaaz1584.6Sci Adv
ARTICLE TOOLS http://advances.sciencemag.org/content/6/23/eaaz1584
MATERIALS
SUPPLEMENTARY http://advances.sciencemag.org/content/suppl/2020/06/01/6.23.eaaz1584.DC1
REFERENCES http://advances.sciencemag.org/content/6/23/eaaz1584#BIBL
This article cites 39 articles, 3 of which you can access for free
PERMISSIONS http://www.sciencemag.org/help/reprints-and-permissions
Terms of ServiceUse of this article is subject to the
is a registered trademark of AAAS.Science AdvancesYork Avenue NW, Washington, DC 20005. The title
(ISSN 2375-2548) is published by the American Association for the Advancement of Science, 1200 NewScience Advances
BY).
Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC
Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of
on June 5, 2020http://advances.sciencemag.org/Downloaded from
... The relative impairment of glutamate clearance by astrocytes in FHM2 mice is activity dependent, being larger after a train of pulses than after a single pulse stimulation and increasing with increasing stimulation frequency in cortical slices [101]. A reduced rate of glutamate clearance was also found in the cingulate cortex of FHM2 mice [124]. The expression of glutamate transporter GLAST, which appears implicated in astrocyte-mediated glutamate uptake in this cortical region [125], is reduced in the cingulate cortex of the FHM2 mutants [124]. ...
... A reduced rate of glutamate clearance was also found in the cingulate cortex of FHM2 mice [124]. The expression of glutamate transporter GLAST, which appears implicated in astrocyte-mediated glutamate uptake in this cortical region [125], is reduced in the cingulate cortex of the FHM2 mutants [124]. In both somatosensory and cingulate cortices, also the rate of clearance of K + ions released during neuronal activity is reduced in FHM2 mice [101] [124], showing the active role of the α2 NKA in K + ions clearance. ...
... Besides increased susceptibility to CSD, another key migraine relevant phenotype shown by FHM2 mice is increased nociceptive response to facial mechanical stimulation after systemic injection of the nitric oxide donor nitroglycerin (NTG) [124]. In migraineurs, NTG administration induces a delayed migraine-like headache with associated features such as premonitory symptoms and allodynia [139,140]. ...
Article
Full-text available
Glutamate levels and lifetime in the brain extracellular space are dinamically regulated by a family of Na⁺- and K⁺-dependent glutamate transporters, which thereby control numerous brain functions and play a role in numerous neurological and psychiatric diseases. Migraine is a neurological disorder characterized by recurrent attacks of typically throbbing and unilateral headache and by a global dysfunction in multisensory processing. Familial hemiplegic migraine type 2 (FHM2) is a rare monogenic form of migraine with aura caused by loss-of-function mutations in the α2 Na/K ATPase (α2NKA). In the adult brain, this pump is expressed almost exclusively in astrocytes where it is colocalized with glutamate transporters. Knockin mouse models of FHM2 (FHM2 mice) show a reduced density of glutamate transporters in perisynaptic astrocytic processes (mirroring the reduced expression of α2NKA) and a reduced rate of glutamate clearance at cortical synapses during neuronal activity and sensory stimulation. Here we review the migraine-relevant alterations produced by the astrocytic glutamate transport dysfunction in FHM2 mice and their underlying mechanisms, in particular regarding the enhanced brain susceptibility to cortical spreading depression (the phenomenon that underlies migraine aura and can also initiate the headache mechanisms) and the enhanced algesic response to a migraine trigger.
... In FHM2 mutants, reduced membrane expression of α2 NKA in the cortical astrocytes led to decreased rates of glutamate and k + clearance, which, in turn, promotes CSD induced neuronal migraine headache (Capuani et al., 2016). Recently, studies further revealed deficient glutamate uptake of astrocytes in the cingulate cortex from FHM2 mutant mice, in which excessive glutamatergic transmission resulted in enhanced cortical excitability by inducing N-methyl-D-aspartate (NMDA) receptor-mediated transmission in the cingulate cortex and somatosensory cortex pyramidal neurons, ultimately increased sensitivity and promote migraine-like cranial pain states (Romanos et al., 2020;Romanos et al., 2019) (Fig. 5A). ...
Article
Chronic pain is a maladaptive condition affecting 7%- 10% of the population worldwide and can be accompanied by depression, anxiety, and insomnia. In particular, chronic pain is becoming more common due to the increasing incidence of diabetes mellitus, cancer, systemic (body-wide) autoimmune, trauma, and infections that attack nerve tissues with an aging global population. Upon stimuli, pain responses are evoked from nociceptive primary sensory neurons in the peripheral nervous system (PNS). Still, pathological changes leading to central sensitization of the pain circuitry in the central nervous system (CNS) is a key mechanism underlying pain maintenance. In humans, chronic pain can last for years, even after the observable signs and symptoms of the primary inflammation or damage have resolved. It is clear that astrocytes, the most abundant cell type in the CNS, are highly involved in regulating pain signaling under health and disease. Multiple astrocyte subsets and diversified activation states driven by intrinsic and extrinsic cues have recently been identified in the spinal cord and brain, playing complex roles in pain development and resolution. Targeting detrimental astrocyte subtypes and activity is considered a promising pain management strategy. Here, we integrate the latest findings to review differential astrocytes activities in distinct regions of the CNS during pain pathophysiology and discuss the underlying molecular mechanisms that control their mode of action in beneficial or/and harmful aspects of pain. Finally, we provide a translational overview of current progress for pain therapies via modulating astrocytic activity.
... For instance, in CM patients, stronger structural connectivity was found between the caudal anterior cingulate cortex (ACC) and other brain regions [5,10] and the N-acetylaspartate of bilateral thalami and right anterior cingulate decreased [6]. In the familial hemiplegic migraine type 2 (FHM2) mouse model, migraine-relevant hypersensitivity triggered by NTG has been attributed to altered neural function in the cingulate cortex [45] and CGRP receptors distributed in the cingulate cortex [39]. However, the ACC did not show high c-Fos activity in LEV-induced migraine mice. ...
Preprint
Full-text available
Background Chronic migraine is a common and disabling disorder. Functional MRI has established that abnormal brain region activation is present in chronic migraine. Drugs targeting the calcitonin gene-related peptide (CGRP) or its receptor have been reported to be efficient for treating chronic migraine. The CGRP signaling pathway has been documented in two types of preclinical migraine mouse models. However, it remains unclear how an active specific brain region develops migraine-like pain and whether CGRP receptor antagonists can alter specific brain region activation and relieve migraine-like pain. Therefore, we sought to investigate brain activation and the effect of olcegepant treatment on brain activation in two chronic migraine models and provide a reference for future research on neural circuits. Methods Repeated administration of nitroglycerin (NTG) or levcromakalim(LEV) was conducted to establish two types of preclinical migraine mouse models to stimulate human migraine-like pain. Mechanical hypersensitivity was evaluated using the von Frey filament test. Then, we evaluated the activation of different brain regions using c-Fos and NeuN staining. Olcegepant, a CGRP receptor-specific antagonist, was administered to explore its countering effect on brain region activation and mechanical hyperalgesia. Results After treatment with NTG and LEV, acute and chronic basal mechanical hyperalgesia was observed in the migraine models. Olcegepant, a CGRP receptor selective antagonist, significantly alleviated mechanical hyperalgesia in both models. In NTG-induced chronic migraine mice, the medial prefrontal cortex (mPFC), anterior cingulate cortex (ACC), and caudal part of the spinal trigeminal nucleus (Sp5c) showed a significant increase in c-Fos expression, while olcegepant reduced c-Fos expression. No change in c-Fos expression was found in the paraventricular thalamic nucleus (PVT) and ventrolateral periaqueductal gray (vlPAG). In LEV-induced migraine mice, mPFC, PVT and Sp5c showed a significant increase in c-Fos expression and olcegepant reduced c-Fos expression. No change in c-Fos expression was found in vlPAG and ACC. Conclusions Our study demonstrated activation of the medial prefrontal cortex and caudal part of the spinal trigeminal nucleus in both chronic migraine models. Olcegepant may alleviate hyperalgesia of the hind paw and periorbital area by attenuating brain activation in chronic migraine.
... Interestingly, FHM2 knock-in mice, having reduced rate of glutamate clearance at cortical excitatory synapses (Leo et al., 2011;Capuani et al., 2016;Parker et al., 2021), show facilitation of NMDA spikes in L5 PC tuft dendrites in cingulate cortex slices, which is correlated with enhanced sensitivity to a migrainerelevant head pain trigger (Romanos et al., 2020); they also show enhanced activation of extrasynaptic GluN1-N2B NMDA receptors in L2/3 PC apical dendrites in barrel cortex slices, whose inhibition rescues the facilitation of experimental CSD (Crivellaro et al., 2021). In vivo, opposite effects of FHM1 mutations on the generation of NMDA and Ca spikes are predicted on the basis of the enhanced FDDI in FHM1 mice (Murayama et al., 2009) and the predicted enhanced excitatory transmission at the L1 topdown synaptic inputs and enhanced activation of the disinhibitory VIP IN-SOM IN-PC microcircuit. ...
Article
Migraine is a complex brain disorder, characterized by attacks of unilateral headache and global dysfunction in multisensory information processing, whose underlying cellular and circuit mechanisms remain unknown. The finding of enhanced excitatory, but unaltered inhibitory, neurotransmission at cortical synapses between pyramidal cells (PCs) and fast-spiking interneurons (FS INs) in mouse models of familial hemiplegic migraine (FHM) suggested the hypothesis that dysregulation of the excitatory-inhibitory (E/I) balance in specific circuits is a key pathogenic mechanism. Here, we investigated the cortical layer 2/3 (L2/3) feedback inhibition microcircuit involving somatostatin-expressing (SOM) INs in FHM1 mice of both sexes carrying a gain-of-function mutation in CaV2.1. Unitary inhibitory neurotransmission at SOM IN-PC synapses was unaltered while excitatory neurotransmission at both PC-SOM IN and PC-PC synapses was enhanced, because of increased probability of glutamate release, in FHM1 mice. Short-term synaptic depression was enhanced at PC-PC synapses while short-term synaptic facilitation was unaltered at PC-SOM IN synapses during 25-Hz repetitive activity. The frequency-dependent disynaptic inhibition (FDDI) mediated by SOM INs was enhanced, lasted longer and required shorter high-frequency bursts to be initiated in FHM1 mice. These findings, together with previous evidence of enhanced disynaptic feedforward inhibition by FS INs, suggest that the increased inhibition may effectively counteract the increased recurrent excitation in FHM1 mice and may even prevail in certain conditions. Considering the involvement of SOM INs in γ oscillations, surround suppression and context-dependent sensory perception, the facilitated recruitment of SOM INs, together with the enhanced recurrent excitation, may contribute to dysfunctional sensory processing in FHM1 and possibly migraine.SIGNIFICANCE STATEMENTMigraine is a complex brain disorder, characterized by attacks of unilateral headache and global dysfunction in multisensory information processing, whose underlying cellular and circuit mechanisms remain unknown, although dysregulation of the excitatory-inhibitory (E/I) balance in specific circuits could be a key pathogenic mechanism. Here, we provide insights into these mechanisms by investigating the cortical feedback inhibition microcircuit involving somatostatin-expressing interneurons (SOM INs) in a mouse model of a rare monogenic migraine. Despite unaltered inhibitory synaptic transmission, the disynaptic feedback inhibition mediated by SOM INs was enhanced in the migraine model because of enhanced recruitment of the INs. Recurrent cortical excitation was also enhanced. These alterations may contribute to context-dependent sensory processing dysfunctions in migraine.
... Astrocytes are essential for normal neuronal functions, and there is emerging evidence for their role in migraine pathogenesis [85,86]. So far, there has not been any report associating astrocytes, CGRP, epigenetics, and migraine, but it was shown that CGRP induced the acetylation of H3K9 in astrocytes linked with neuroinflammation in rats with neuropathic pain [87]. ...
Article
Full-text available
The calcitonin gene-related peptide (CGRP) is implicated in the pathogenesis of several pain-related syndromes, including migraine. Targeting CGRP and its receptor by their antagonists and antibodies was a breakthrough in migraine therapy, but the need to improve efficacy and limit the side effects of these drugs justify further studies on the regulation of CGRP in migraine. The expression of the CGRP encoding gene, CALCA, is modulated by epigenetic modifications, including the DNA methylation, histone modification, and effects of micro RNAs (miRNAs), circular RNAs, and long-coding RNAs (lncRNAs). On the other hand, CGRP can change the epigenetic profile of neuronal and glial cells. The promoter of the CALCA gene has two CpG islands that may be specifically methylated in migraine patients. DNA methylation and lncRNAs were shown to play a role in the cell-specific alternative splicing of the CALCA primary transcript. CGRP may be involved in changes in neural cytoarchitecture that are controlled by histone deacetylase 6 (HDAC6) and can be related to migraine. Inhibition of HDAC6 results in reduced cortical-spreading depression and a blockade of the CGRP receptor. CGRP levels are associated with the expression of several miRNAs in plasma, making them useful peripheral markers of migraine. The fundamental role of CGRP in inflammatory pain transmission may be epigenetically regulated. In conclusion, epigenetic connections of CGRP should be further explored for efficient and safe antimigraine therapy.
... Finally, other mechanisms than intrinsic and synaptic plasticity, such as network rearrangements or altered functional connectivity, can account for changes in functional neuronal activity. Thus, stimulusevoked firing of layer V pyramidal neurons is enhanced (i) in ACC of neuropathic mice, following down-regulation of hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels in dendrites, thus facilitating the integration of excitatory inputs (Santello and Nevian, 2015), (ii) in S1 of neuropathic mice, after concomitant reductions in somatostatin-expressing and parvalbumin-expressing inhibitory neuronal activities (Cichon et al., 2017) and, (iii) in ACC of mice in which reduced expression of astrocytic Na + , K + -ATPases impairs astrocytic glutamate uptake (Romanos et al., 2020). Finally, facilitated layer II-III pyramidal neuronal output can enhance layer V pyramidal neuronal activity, as facilitated S1 output enhances ACC activity under chronic inflammatory conditions (Eto et al., 2011). ...
Article
Patients with chronic pain, especially orofacial pain, often suffer from affective disorders, including anxiety. Previous studies largely focused on the role of the caudal anterior cingulate cortex (cACC) in affective responses to pain, long-term potentiation (LTP) in cACC being thought to mediate the interaction between anxiety and chronic pain. But recent evidence indicates that the rostral ACC (rACC), too, is implicated in processing affective pain. However, whether such processing is associated with neuronal and/or synaptic plasticity is still unknown. We addressed this issue in a chronic facial inflammatory pain model (complete Freund's adjuvant model) in rats, by combining behavior, Fos protein immunochemistry and ex vivo intracellular recordings in rACC slices prepared from these animals. Facial mechanical allodynia occurs immediately after CFA injection, peaks at post-injection day 3 and progressively recovers until post-injection days 10-11, whereas anxiety is delayed, being present at post-injection day 10, when sensory hypersensitivity is relieved, but, notably, not at post-injection day 3. Fos expression reveals that neuronal activity follows a bi-phasic time course in bilateral rACC: first enhanced at post-injection day 3, it gets strongly depressed at post-injection day 10. Ex vivo recordings from lamina V pyramidal neurons, the rACC projecting neurons, show that both their intrinsic excitability and excitatory synaptic inputs have undergone long-term depression (LTD) at post-injection day 10. Thus chronic pain processing is associated with dynamic changes in rACC activity: first enhanced and subsequently decreased, at the time of anxiety-like behavior. Chronic pain-induced anxiety might thus result from a rACC deactivation-cACC hyperactivation interplay.
... In parallel, this study also shows that local rescue of the impairment in glutamate uptake in ACC can prevent abnormal NMDA spikes and reduces sensitivity to cranial pain triggers in migraine mice. 166 Recently, we found that cortical plasticity contributes to periorbital allodynia and anxiety in rats suffering chronic migraine, in which both glutamate release and NMDAR responses in ACC are increased (unpublished data). Furthermore, our recent slice experiments have shown that CGRP induces a long-term change of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor-mediated responses in the ACC and insula. ...
Article
Full-text available
Migraine is the second most prevalent disorder in the world; yet, its underlying mechanisms are still poorly understood. Cumulative studies have revealed pivotal roles of cerebral cortex in the initiation, propagation, and termination of migraine attacks as well as the interictal phase. Investigation of basic mechanisms of the cortex in migraine not only brings insight into the underlying pathophysiology but also provides the basis for designing novel treatments. We aim to summarize the current research literatures and give a brief overview of the cortex and its role in migraine, including the basic structure and function; structural, functional, and biochemical neuroimaging; migraine-related genes; and theories related to cortex in migraine pathophysiology. We propose that long-term plasticity of synaptic transmission in the cortex encodes migraine.
Article
Full-text available
Background: Chronic migraine is a common and highly disabling disorder. Functional MRI has indicated that abnormal brain region activation is linked with chronic migraine. Drugs targeting the calcitonin gene-related peptide (CGRP) or its receptor have been reported to be efficient for treating chronic migraine. The CGRP signaling was also shared in two types of chronic migraine models (CMMs). However, it remains unclear whether the activation of specific brain regions could contribute to persistent behavioral sensitization, and CGRP receptor antagonists relieve migraine-like pain in CMMs by altering specific brain region activation. Therefore, it's of great interest to investigate brain activation pattern and the effect of olcegepant (a CGRP receptor-specific antagonist) treatment on alleviating hyperalgesia by altering brain activation in two CMMs, and provide a reference for future research on neural circuits. Methods: Repeated administration of nitroglycerin (NTG) or levcromakalim (LEV) was conducted to stimulate human migraine-like pain and establish two types of CMMs in mice. Mechanical hypersensitivity was evaluated by using the von Frey filament test. Then, we evaluated the activation of different brain regions with c-Fos and NeuN staining. Olcegepant was administered to explore its effect on mechanical hyperalgesia and brain region activation. Results: In two CMMs, acute and basal mechanical hyperalgesia was observed, and olcegepant alleviated mechanical hyperalgesia. In the NTG-induced CMM, the medial prefrontal cortex (mPFC), anterior cingulate cortex (ACC), and the caudal part of the spinal trigeminal nucleus (Sp5c) showed a significant increase of c-Fos expression in the NTG group (p < 0.05), while pre-treatment with olcegepant reduced c-Fos expression compared with NTG group (p < 0.05). No significant difference of c-Fos expression was found in the paraventricular thalamic nucleus (PVT) and ventrolateral periaqueductal gray (vlPAG) between the vehicle control and NTG group (p > 0.05). In the LEV-induced CMM, mPFC, PVT, and Sp5c showed a significant increase of c-Fos expression between vehicle control and LEV group, and olcegepant reduced c-Fos expression (p < 0.05). No significant difference in c-Fos expression was found in vlPAG and ACC (p > 0.05). Conclusions: Our study demonstrated the activation of mPFC and Sp5c in two CMMs. Olcegepant may alleviate hyperalgesia of the hind paw and periorbital area by attenuating brain activation in CMMs.
Article
The rostral anterior cingulate cortex (rACC) has been found to be an important brain region in mediating visceral hypersensitivity. However, the underlying mechanisms remain unclear. This study aimed to explore the role of astrocytes in the maintenance of visceral hypersensitivity induced by chronic water avoidance stress (WAS) as well as the potential signaling pathway that activates astrocytes in the rACC. We found that ACC-reactive astrogliosis resulted in the overexpression of c-fos, TSP-1, and BDNF in stress-related visceral hypersensitivity rats. Visceral hypersensitivity was reversed by pharmacological inhibition of astrocytic activation after WAS, as were the overexpression of c-fos, TSP-1 and BDNF. Activation of the astrocytic Gi-pathway increased the visceral sensitivity and expression of c-fos, TSP-1, and BDNF. Visceral hypersensitivity was also ameliorated by the pharmacological inhibition of ERK and STAT1 phosphorylation after WAS. Furthermore, inhibition of the ERK-STAT1 cascade reduced astrocytic activation. These findings suggest that astrocytic ERK/STAT1 signaling in the rACC contributes to the maintenance of stress-related visceral hypersensitivity. Perspective Visceral hypersensitivity is a key factor in the pathophysiology of irritable bowel syndrome. This study highlights the important role of astrocytic ERK/STAT1 signaling in activating astrocytes in the rostral anterior cingulate cortex, which contributes to visceral hypersensitivity.
Article
Reactive astrocytes are commonly activated in the spinal dorsal horn (SDH) of various animal models of pathological pain. Previous investigations suggest an association between astrogliosis and pain pathogenesis. However, our understanding of the mechanisms underlying astrogliosis activation and the contributions of reactive astrocytes to pain neural circuit malfunction is rudimentary. This short review highlights recent advances in these areas.
Article
Full-text available
Light-sheet microscopy is an ideal technique for imaging large cleared samples; however, the community is still lacking instruments capable of producing volumetric images of centimeter-sized cleared samples with near-isotropic resolution within minutes. Here, we introduce the mesoscale selective plane-illumination microscopy initiative, an open-hardware project for building and operating a light-sheet microscope that addresses these challenges and is compatible with any type of cleared or expanded sample (www.mesospim.org).
Article
Full-text available
Removal of synaptically-released glutamate by astrocytes is necessary to spatially and temporally limit neuronal activation. Recent evidence suggests that astrocytes may have specialized functions in specific circuits, but the extent and significance of such specialization are unclear. By performing direct patch-clamp recordings and two-photon glutamate imaging, we report that in the somatosensory cortex, glutamate uptake by astrocytes is slower during sustained synaptic stimulation when compared to lower stimulation frequencies. Conversely, glutamate uptake capacity is increased in the frontal cortex during higher frequency synaptic stimulation, thereby limiting extracellular buildup of glutamate and NMDA receptor activation in layer 5 pyramidal neurons. This efficient glutamate clearance relies on Na⁺/K⁺-ATPase function and both GLT-1 and non-GLT-1 transporters. Thus, by enhancing their glutamate uptake capacity, astrocytes in the frontal cortex may prevent excessive neuronal excitation during intense synaptic activity. These results may explain why diseases associated with network hyperexcitability differentially affect individual brain areas.
Article
Full-text available
The identity of cortical circuits mediating nociception and pain is largely unclear. The cingulate cortex is consistently activated during pain, but the functional specificity of cingulate divisions, the roles at distinct temporal phases of central plasticity and the underlying circuitry are unknown. Here we show in mice that the midcingulate division of the cingulate cortex (MCC) does not mediate acute pain sensation and pain affect, but gates sensory hypersensitivity by acting in a wide cortical and subcortical network. Within this complex network, we identified an afferent MCC–posterior insula pathway that can induce and maintain nociceptive hypersensitivity in the absence of conditioned peripheral noxious drive. This facilitation of nociception is brought about by recruitment of descending serotonergic facilitatory projections to the spinal cord. These results have implications for our understanding of neuronal mechanisms facilitating the transition from acute to long-lasting pain.
Article
Full-text available
Migraine is a common disabling brain disorder. A subtype of migraine with aura (familial hemiplegic migraine type 2: FHM2) is caused by loss-of-function mutations in α2 Na(+),K(+) ATPase (α2 NKA), an isoform almost exclusively expressed in astrocytes in adult brain. Cortical spreading depression (CSD), the phenomenon that underlies migraine aura and activates migraine headache mechanisms, is facilitated in heterozygous FHM2-knockin mice with reduced expression of α2 NKA The mechanisms underlying an increased susceptibility to CSD in FHM2 are unknown. Here, we show reduced rates of glutamate and K(+) clearance by cortical astrocytes during neuronal activity and reduced density of GLT-1a glutamate transporters in cortical perisynaptic astrocytic processes in heterozygous FHM2-knockin mice, demonstrating key physiological roles of α2 NKA and supporting tight coupling with GLT-1a. Using ceftriaxone treatment of FHM2 mutants and partial inhibition of glutamate transporters in wild-type mice, we obtain evidence that defective glutamate clearance can account for most of the facilitation of CSD initiation in FHM2-knockin mice, pointing to excessive glutamatergic transmission as a key mechanism underlying the vulnerability to CSD ignition in migraine.
Article
Introduction: Familial hemiplegic migraine 2 is a pathology linked to mutation of the ATP1A2 gene producing loss of function of the α2 Na+/K+-ATPase (NKA). W887R/+ knock-in (KI) mice are used to model the familial hemiplegic migraine 2 condition and are characterized by 50% reduced NKA expression in the brain and reduced rate of K+ and glutamate clearance by astrocytes. These alterations might, in turn, produce synaptic changes in synaptic transmission and plasticity. Memory and learning deficits observed in familial hemiplegic migraine patients could be ascribed to a possible alteration of hippocampal neuronal plasticity and measuring possible changes of long-term potentiation in familial hemiplegic migraine 2 KI mice might provide insights to strengthen this link. Results: Here we have investigated synaptic plasticity in distinct hippocampal regions in familial hemiplegic migraine 2 KI mice. We show that the dentate gyrus long-term potentiation of familial hemiplegic migraine 2 mice is abnormally increased in comparison with control animals. Conversely, in the CA1 area, KI and WT mice express long-term potentiation of similar amplitude. Conclusions: The familial hemiplegic migraine 2 KI mice show region-dependent hippocampal plasticity abnormality, which might underlie some of the memory deficits observed in familial migraine.
Article
Astrocytes serve important roles that affect recruitment and function of neurons at the local and network levels. Here we review the contributions of astrocyte signaling to synaptic plasticity, neuronal network oscillations, and memory function. The roles played by astrocytes are not fully understood, but astrocytes seem to contribute to memory consolidation and seem to mediate the effects of vigilance and arousal on memory performance. Understanding the role of astrocytes in cognitive processes may also advance our understanding of how these processes go awry in pathological conditions. Indeed, abnormal astrocytic signaling can cause or contribute to synaptic and network imbalances, leading to cognitive impairment. We discuss evidence for this from animal models of Alzheimer’s disease and multiple sclerosis and from animal studies of sleep deprivation and drug abuse and addiction. Understanding the emerging roles of astrocytes in cognitive function and dysfunction will open up a large array of new therapeutic opportunities.
Article
GLT-1, the major glutamate transporter, is expressed at perisynaptic astrocytic processes (PAP) and axon terminals (AxT). GLT-1 is coupled to Na+/K+-ATPase (NKA) α1-3 isoforms, whose subcellular distribution and spatial organization in relationship to GLT-1 are largely unknown. Using several microscopy techniques, we showed that at excitatory synapses α1 and α3 are exclusively neuronal (mainly in dendrites and in some AxT), while α2 is predominantly astrocytic. GLT-1 displayed a differential colocalization with α1-3. GLT-1/α2 and GLT-1/α3 colocalization was higher in GLT-1 positive puncta partially (for GLT-1/α2) or almost totally (for GLT-1/α3) overlapping with VGLUT1 positive terminals than in nonoverlapping ones. GLT-1 colocalized with α2 at PAP, and with α1 and α3 at AxT. GLT-1 and α2 gold particles were ∼1.5-2 times closer than GLT-1/α1 and GLT-1/α3 particles. GLT-1/α2 complexes (edge to edge interdistance of gold particles ≤50 nm) concentrated at the perisynaptic region of PAP membranes, whereas neuronal GLT-1/α1 and GLT-1/α3 complexes were fewer and more uniformly distributed in AxT. These data unveil different composition of GLT-1 and α subunits complexes in the glial and neuronal domains of excitatory synapses. The spatial organization of GLT-1/α1-3 complexes suggests that GLT-1/NKA interaction is more efficient in astrocytes than in neurons, further supporting the dominant role of astrocytic GLT-1 in glutamate homeostasis.
Article
Migraine is an extremely common but poorly understood nervous system disorder. We conceptualize migraine as a disorder of sensory network gain and plasticity, and we propose that this framing makes it amenable to the tools of current systems neuroscience. Migraine affects 12% of the world's population yet remains poorly understood. Brennan and Pietrobon propose that migraine can be approached as a disorder of sensory gain and plasticity, making it amenable to the tools and insights of current systems neuroscience.
Article
Localized, heterogeneous calcium transients occur throughout astrocytes, but the characteristics and long-term stability of these signals, particularly in response to sensory stimulation, remain unknown. Here, we used a genetically encoded calcium indicator and an activity-based image analysis scheme to monitor astrocyte calcium activity in vivo. We found that different subcellular compartments (processes, somata, and endfeet) displayed distinct signaling characteristics. Closer examination of individual signals showed that sensory stimulation elevated the number of specific types of calcium peaks within astrocyte processes and somata, in a cortical layer-dependent manner, and that the signals became more synchronous upon sensory stimulation. Although mice genetically lacking astrocytic IP3R-dependent calcium signaling (Ip3r2−/−) had fewer signal peaks, the response to sensory stimulation was sustained, suggesting other calcium pathways are also involved. Long-term imaging of astrocyte populations revealed that all compartments reliably responded to stimulation over several months, but that the location of the response within processes may vary. These previously unknown characteristics of subcellular astrocyte calcium signals provide new insights into how astrocytes may encode local neuronal circuit activity.
Article
The anterior cingulate cortex (ACC) is activated in both acute and chronic pain. In this Review, we discuss increasing evidence from rodent studies that ACC activation contributes to chronic pain states and describe several forms of synaptic plasticity that may underlie this effect. In particular, one form of long-term potentiation (LTP) in the ACC, which is triggered by the activation of NMDA receptors and expressed by an increase in AMPA-receptor function, sustains the affective component of the pain state. Another form of LTP in the ACC, which is triggered by the activation of kainate receptors and expressed by an increase in glutamate release, may contribute to pain-related anxiety.