Published in Volume
119, Issue 1
(January 5, 2009)J Clin Invest.
Copyright © 2009, American Society for Clinical
Genetic and hormonal factors modulate spreading depression and transient
hemiparesis in mouse models of familial hemiplegic migraine type 1
1Stroke and Neurovascular Regulation Laboratory, Department of Radiology,
Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA.
2Department of Neurology, University of Duisburg-Essen, Essen,
3Department of Neurology, University of Texas Medical School at
Houston, Houston, Texas, USA.
4Department of Biology, Boston University,
Boston, Massachusetts, USA.
5Department of Neurology and
6Department of Human Genetics, Leiden University Medical Center, Leiden,
7Stroke Service and Neuroscience Intensive Care Unit,
Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston,
Address correspondence to: Cenk Ayata, Stroke and Neurovascular Regulation
Laboratory, Massachusetts General Hospital, 149 13th Street, Room 6403, Charlestown,
Massachusetts 02129, USA. Phone: (617) 726-8021; Fax: (617) 726-2547; E-mail:
First published December 22, 2008
Received for publication April 28,
2008, and accepted in revised form October 8,
Familial hemiplegic migraine type 1 (FHM1) is an autosomal dominant subtype of
migraine with aura that is associated with hemiparesis. As with other types of
migraine, it affects women more frequently than men. FHM1 is caused by mutations in
the CACNA1A gene, which encodes the α1A
subunit of Cav2.1 channels; the R192Q mutation in CACNA1A
causes a mild form of FHM1, whereas the S218L mutation causes a severe, often lethal
phenotype. Spreading depression (SD), a slowly propagating neuronal and glial cell
depolarization that leads to depression of neuronal activity, is the most likely
cause of migraine aura. Here, we have shown that transgenic mice expressing R192Q or
S218L FHM1 mutations have increased SD frequency and propagation speed; enhanced
corticostriatal propagation; and, similar to the human FHM1 phenotype, more severe
and prolonged post-SD neurological deficits. The susceptibility to SD and
neurological deficits is affected by allele dosage and is higher in S218L than R192Q
mutants. Further, female S218L and R192Q mutant mice were more susceptible to SD and
neurological deficits than males. This sex difference was abrogated by ovariectomy
and senescence and was partially restored by estrogen replacement, implicating
ovarian hormones in the observed sex differences in humans with FHM1. These findings
demonstrate that genetic and hormonal factors modulate susceptibility to SD and
neurological deficits in FHM1 mutant mice, providing a potential mechanism for the
phenotypic diversity of human migraine and aura.
Spreading depression (SD) is characterized by an intense depolarization of neuronal and
glial membranes that propagates in brain tissue at a rate of approximately 3 mm/min
Evoked when local extracellular K+ concentrations exceed a critical
threshold, SD is associated with disruption of membrane ionic gradients, and massive
K+ efflux and glutamate release; both are believed to depolarize adjacent
neurons and glia, thereby facilitating its spread. There is growing evidence from animal
experiments, suggesting that cortical SD (CSD) is the electrophysiological event
underlying migraine aura and a possible trigger of headache mechanisms (3–5). In humans, evidence for a causal role of SD in the aura comes from
functional MRI performed during migraine with aura attacks (6, 7).
SD is also implicated in the pathophysiology of familial hemiplegic migraine (FHM), an
autosomal dominant subtype of migraine with aura associated with hemiparesis (8). The aura and headache features are otherwise
identical to those in the common, multifactorial forms of migraine. This and the fact
that the majority of FHM patients also experience attacks of common migraine with or
without aura (9) make FHM a valid model for
studying the pathogenesis of common, complex types of migraine.
Thus far, 3 FHM genes have been identified (10).
The FHM1 CACNA1A gene encodes the pore-forming
α1A-subunit of neuronal, voltage-gated Cav2.1
(previously known as P/Q-type) calcium channels (11, 12). Two knock-in FHM1 mouse models,
carrying the human pathogenic R192Q or S218L missense mutation, were generated (13, 14). In
patients, the R192Q mutation causes pure FHM without other associated neurological
features (12), whereas the S218L mutation causes
a severe migraine phenotype with excessive and often fatal cerebral edema (15). When expressed in transfected cultured neurons,
both mutations shift channel opening toward more negative membrane potentials and delay
channel inactivation; the S218L mutation causes more pronounced single-channel gain of
function than R192Q (14, 16). As a result, channels open with smaller depolarization and stay
open longer, allowing more Ca2+ to enter presynaptic terminals. FHM1 mouse
models exhibit a reduced threshold for electrically evoked CSD and increased SD velocity
Gonadal hormones are important modulators of migraine (18) and cortical excitability (19–21). In migraine with
aura, the incidence of attacks reportedly increases during periods of high circulating
estrogen (18, 22, 23). Higher plasma estrogen
concentrations were measured during normal menstrual cycle in migraineurs with aura
(24). Whereas the prevalence of migraine is
similar in boys and girls before puberty (4%), after puberty it rises to about 3-fold
higher in adult females (25%) than in males (8%) (25, 26). A female preponderance is also
described for familial (5:2 ratio) and sporadic (4.25:1 ratio) hemiplegic migraine
In this study, we investigated the modulating effect of different allelic mutations,
gene dosage, and gonadal hormones on SD susceptibility and subsequent neurological
deficits as surrogates for migraine aura in mouse models of FHM1. We found that both
FHM1 mutant strains exhibited enhanced SD susceptibility, and SD induced severe and
prolonged unilateral motor deficits akin to the human FHM phenotype. Furthermore, both
SD susceptibility and severity of neurological deficits were modulated by: (a) the
degree of gain of function caused by allelic mutations, (b) allele dosage, and (c)
female gonadal hormones. Our data provide a potential mechanism for the severe and
prolonged neurological deficits in FHM patients and underscore that genetic and hormonal
factors contribute to the phenotypic diversity of human migraine syndromes.
CSD susceptibility in FHM1 mutant mice is modulated by Cav2.1 gain of
function, allele dosage, and ovarian hormones. SD susceptibility was assessed by: (a) counting the number of evoked SDs during
continuous topical KCl (300 mM) application and (b) measuring the propagation speed
of SDs between 2 recording electrodes. The values in the different strains were
compared. In WT mice, epidural KCl evoked repetitive CSDs (9.5 ± 1.0
SDs/h) with a propagation speed of 2.8 ± 0.2 mm/min. Both the frequency
and the propagation speed were increased in the R192Q mutants and even more so in the
S218L mutants (Figure 1). Furthermore,
heterozygous mice of both FHM1 mutant strains showed SD frequencies and propagation
speeds intermediate between those in WT and homozygous mutant mice, indicating an
allele dosage effect. The duration or amplitude of SDs after KCl application did not
differ among the groups (Table 1). The
electrocorticogram did not show seizure activity in mutant mice during these SD
recordings under anesthesia.
Enhanced CSD susceptibility in FHM1 mutant mice. (A) Representative electrophysiological recordings showing increased
frequency of slow (DC) potential shifts during repetitive CSDs in male and female
homozygous R192Q or S218L mutants compared with WT mice evoked by topical KCl
application (300 mM) for 30 minutes. The number of CSDs was substantially higher
in FHM1 mutant strains compared with WT controls and in female mutants versus
males. Calibration bars: vertical, 20 mV; horizontal, 10 minutes. (B)
The impact of R192Q and S218L mutations, allele dosage, and sex on CSD frequency
and propagation speed (upper and lower graphs, respectively). Higher CSD
frequencies and propagation speeds were found in both FHM1 mutants, with values
higher in S218L than in R192Q mutants and an allele dosage relation. CSD frequency
was greater and propagation faster in females compared with males in both FHM1
mutant strains. No sex difference was found in WT mice. Covariance analysis
revealed that 82% of variation in CSD frequency and 93% of variation in CSD
propagation speed were explained by the independent variables mutation, genotype,
and sex (see Methods). Numbers of mice per group are shown within the bars. HET,
heterozygous; HOM, homozygous. Data are mean ± standard deviation.
**P < 0.001, *P < 0.01 versus
male in CSD frequency and propagation speed;
†P < 0.001 versus WT and homozygous
mutant; ‡P < 0.001 versus WT and
heterozygous mutant; §P < 0.001
versus corresponding R192Q genotype.
Electrophysiological measures of CSD and systemic physiological parameters in FHM1
SD susceptibility (i.e., frequency and propagation speed) was strikingly higher in
females than in males of both FHM1 mutant strains but not in WT mice (Figure 1). When tested in R192Q mutant mice, the sex
difference was abrogated by ovariectomy, implicating effects of female gonadal
hormones in adult brain, rather than a perinatal organizational effect of gonadal
hormones or chromosomal sex per se, as modulators of cortical excitability (Figure
2). Consistent with these findings, SD
frequencies and propagation speeds were significantly reduced in senescent female
R192Q mutant mice after putative cessation of estrous cycling and reduced gonadal
hormone production (Figure 2). Chronic estrogen
replacement by subcutaneous implantation of pellets containing 0.075 mg
17β-estradiol (3-week release) was associated with a small increase in SD
susceptibility in ovariectomized R192Q mutant, but not in WT, mice (13 ±
1 vs. 16 ± 1 SDs/h in control and estrogen-treated homozygous R192Q
mutant, n = 5 and 6, respectively; P <
0.01). A lower dose of estrogen (0.025 mg/pellet) was ineffective in both WT and
homozygous R192Q mutant mice (data not shown). Neither gonadectomy nor advanced age
influenced SD susceptibility in WT mice using the described protocols (Figure 2). These data suggest that female gonadal hormones
modulate SD susceptibility only in mice that have a genetic predisposition.
Gonadal hormone–mediated modulation of CSD susceptibility in
female FHM1 mutant mice. The impact of ovariectomy and senescence on CSD susceptibility in WT and
homozygous R192Q mutant mice. Both ovariectomy (3–4 months of age;
gray) and senescence (13 months of age; white) reduced CSD frequency (left panel)
and propagation speed (right panel) in homozygous female R192Q to the level of
male mutants (see Figure 1B). Ovariectomy
(n = 5) and aging (n = 7) had similar effects
in heterozygous female R192Q mice (data not shown). In WT mice, gonadectomy or
senescence did not alter CSD susceptibility. Experiments were performed 3 weeks
after ovariectomy. Numbers of mice for each group are shown within the bars. Ovx,
ovariectomized. Data are mean ± standard deviation.
*P < 0.001, #P < 0.01
versus WT; †P < 0.001 versus naive
young female mice of the same genotype.
FHM1 mutant mice develop severe and prolonged motor deficits after SD that are
modulated by gene dosage and sex. Prior to SD induction, both WT and FHM1 mutant strains appeared phenotypically normal
and did not exhibit neurological deficits when assessed using the wire grip test and
neurological examination protocols. In WT mice, a single SD induced by brief topical
application of 300 mM KCl caused only mild deficits that lasted less than 10 minutes
in the wire grip test and only a mild and short-lasting hemiparesis that completely
recovered before the first neurological assessment at 5 minutes after SD induction
(Figure 3A). In contrast, a single SD caused
hemiplegia with leaning and circling in both R192Q and S218L mutant mice (Figure
3, Supplemental Figure 1, and Supplemental
Videos 1–13; supplemental material available online with this article;
10.1172/JCI36059DS1). In the wire grip test, they were initially unable to
move along the wire and fell more often than WT mice. S218L mutants were more
severely impaired, and unlike R192Q mice, some remained unconscious for up to 10
minutes after a single SD. Homozygous FHM1 mutant mice were impaired more than
heterozygotes, indicating an allele dosage relation (Figure 3). Interestingly, FHM1 mutants showed 1 or more transient
episodes of neurological deterioration after full or partial recovery during the 60-
to 80-minute monitoring period (Supplemental Figure 1E).
Prolonged and severe neurological deficits after SD in FHM1 mutant mice. Time course of neurological deficits in WT and FHM1 mutant mice after a single SD
(A) or 9 SDs over 1 hour (B), as assessed by wire
grip score and latency as well as neurological score. (A) Impact of
allelic mutations and allele dosage. In WT mice, deficits were mild and
short-lasting in wire grip test and undetectable by neurological scoring
(triangles). FHM1 mutants developed severe and prolonged deficits
(P < 0.01 versus WT). These deficits were markedly more
severe and prolonged in homozygous S218L (black circles) than in homozygous R192Q
mutants (gray circles; P < 0.01) or heterozygous S218L
mutants (squares; P < 0.01 in wire grip test,
P < 0.05 in neurological scoring). n =
13, 7, 6, and 3 WT, homozygous R192Q, heterozygous S218L, and homozygous S218L
mutant mice, respectively. (B) Sex differences. In WT mice, deficits
were mild and completely recovered within 45 minutes after 9 SDs. In contrast,
R192Q mutant mice developed severe and prolonged deficits that lasted 80 minutes
or more after 9 SDs (P < 0.01 versus WT). Importantly,
female mutant mice (circles) were more severely affected than males (squares;
P < 0.01). Deficits did not differ between WT males and
females; therefore, data were pooled (triangles). n = 15, 6, and
7 WT, male R192Q, and female R192Q mutant mice, respectively. Error bars were
omitted for clarity but were less than 25% of mean.
All 3 homozygous S218L mutant mice developed generalized seizures 45, 55, and 75
minutes after a single SD. Two of the mice died immediately after the seizure due to
respiratory arrest; the third mouse survived the seizure and completely recovered at
80 minutes. Seizures were not observed in heterozygous S218L or homozygous R192Q
mutant mice during recovery from SD. The deficits in heterozygous S218L mutant mice
in the absence of overt seizure activity suggest that the clinical phenotype was most
likely due to SD and not seizure activity.
Compared with a single SD, multiple SDs (9 in 1 hour) caused more severe motor
deficits. In WT mice, deficits in the wire grip test were detectable for 30 minutes
after the last SD (Figure 3B), whereas multiple
SDs caused even more severe deficits in R192Q mutants. Moreover, female R192Q mutant
mice developed more severe and longer-lasting deficits than males (Figure 3B).
Recovery of cortical evoked potentials after SD is not delayed in FHM1 mutant
mice. To test whether prolonged neurological deficits were due to a slower recovery of
cortical electrophysiological function, we recorded somatosensory evoked potentials
in the whisker barrel cortex after a single SD. Evoked potentials were abolished
after SD but reemerged within approximately 2 minutes and completely recovered to
pre-SD baseline levels within 10 minutes (Figure 4A). The recovery rate was identical in WT and R192Q or S218L mutant mice
(Figure 4, A and B). No seizure activity was
observed in these recordings under anesthesia. These data indicated that delayed
electrophysiological recovery of cortical synaptic function was not the cause of
prolonged and severe unilateral motor deficits after CSD in FHM1 mutant mice.
The recovery of whisker pad stimulation–evoked cortical field
potentials. (A) Representative tracings showing field potentials in a
female WT (top), homozygous female R192Q mutant (middle), and heterozygous female
S218L mutant mouse (bottom), evoked by electrical stimulation of the whisker pad
(black triangles) and recorded from the whisker barrel field using extracellular
glass micropipettes (400 μm depth, layer IV). Evoked potentials
ranging from 2 to 4 mV in amplitude at baseline (column a) were abolished upon
arrival of SD at the recording site (column b) and gradually recovered within less
than 10 minutes (columns c–e), as shown in the time course graph in
B. Calibration bars: vertical, 1 mV; horizontal, 20 ms.
(B) The time course of recovery of somatosensory evoked field
potentials (SSEP) after a single SD (time 0).The AUC for each evoked field
potential (mV • ms) was expressed as percent of pre-SD baseline. The
rate of recovery of cortical evoked field potentials did not differ among WT and
FHM1 mutant mice. Recovery of peak amplitudes also did not differ among groups
(data not shown). Numbers of mice are indicated in the graph. Male and female WT
mice did not differ; therefore, pooled data are shown. Error bars were omitted for
Corticostriatal propagation of SD is facilitated in FHM1 mutant mice. CSD occasionally propagates into subcortical structures in rodent brain (29, 30).
We therefore tested whether prolonged post-SD neurological deficits such as
hemiplegia and circling were associated with enhanced subcortical spread into the
striatum. In WT mice, CSDs did not reach the striatum (Figure 5; see Methods for experimental conditions). In contrast, CSDs
readily propagated into the striatum in FHM1 mutant mice (Figure 5A). Corticostriatal SD propagation was more frequent and arose
with shorter latency in S218L compared with R192Q mutant mice (P
< 0.05), homozygotes compared with heterozygotes of both mutant strains
(P < 0.01), and female homozygous (but not heterozygous)
mutants compared with males (P < 0.01; Figure 5B and Table 2). SD did not propagate to thalamus and hippocampus under our experimental
conditions (n = 7 heterozygous female and homozygous male S218L
mice; n = 2 WT mice; data not shown). Therefore, corticostriatal SD
propagation corresponded well to the severity of post-SD neurological deficits and
was modulated by: (a) the degree of ion channel dysfunction; (b) allele dosage; and
(c) sex. These data implicate corticostriatal SD propagation as a likely explanation
for the more severe motor deficits in FHM1 mutant mice.
Facilitated corticostriatal SD propagation in FHM1 mutant mice. (A) Representative extracellular DC potential shifts recorded
simultaneously from cortex and striatum in female WT and homozygous R192Q and
S218L mutant mice. SD was not observed to propagate into the striatum in any of
the WT mice. A substantial proportion of CSDs propagated into the striatum in
R192Q and to a greater extent in the S218L mutant. Calibration bars: vertical, 20
mV; horizontal, 10 minutes. (B) The frequency of SDs during
continuous topical KCl application (300 mM) to the occipital cortex. CSD frequency
(gray bars) was substantially higher in S218L and to a lesser extent in R192Q
mutant mice than WT. They were higher in females compared with males and
homozygous FHM1 mutants compared with heterozygotes (see Figure 1). CSDs readily propagated into striatum (black
bars) in both FHM1 mutant strains, but never in the WT. Striatal propagation was
more frequent in S218L (lower graphs) compared with R192Q mutants (upper graphs)
and in females (right) compared with males (left), with an allele dosage relation
(see Table 2 for latencies between cortical
and striatal SDs). Covariance analysis revealed that 83% of the variance of
striatal SD frequency was explained by the independent variables mutation,
genotype, and sex (see Methods). n = 3–8 mice per
group as shown within each bar. Data are mean ± standard deviation.
*P < 0.01 versus male;
†P < 0.001,
§P < 0.05 versus heterozygous
mutants; ‡P < 0.01 versus R192Q
Corticostriatal propagation latency of SD
FHM1 mutant mice develop delayed recurrent SDs after a single KCl-induced SD. In order to test whether prolonged neurological deficits were caused by recurrent
SDs, we recorded from cortex and striatum simultaneously for 60 minutes after one
initial SD induced by brief topical KCl application that was followed by extensive
saline wash. In all FHM1 knock-in mice, 1 or more recurrent SDs were observed
throughout the recording period (range, 1–55 minutes after the initial
SD, n = 6 heterozygous female and 2 homozygous male S218L mutants
and n = 3 homozygous female R192Q mutants; Supplemental Figure 3).
None of the WT mice showed SD recurrence. As suggested by their latency, recurrent
SDs appeared to originate from cortex and consistently spread into striatum. In a few
instances, there was evidence for cortico-striato-cortical reentrant SDs. In the
absence of prior SD induction, we did not observe spontaneous SDs during 60-minute
recordings in mutant or WT strains (n = 6).
Migraine susceptibility is modulated by genetic, physiological, and environmental
factors. Here, we provide experimental evidence showing that genetic (Cav2.1
voltage-gated Ca2+ channel mutations) and physiological factors (gonadal
hormones) modulating susceptibility to migraine also modulate SD susceptibility.
Enhanced SD susceptibility shows a clear relation to the degree of single-channel gain
of function caused by S218L and R192Q mutations and is associated with more severe and
prolonged neurological deficits after SD, consistent with the clinical phenotypes
observed in FHM1 patients. Both enhanced SD susceptibility (i.e., frequency and
propagation speed) and neurological deficits are linked to the propensity of
corticostriatal SD propagation and are modulated by the interaction of allelic
mutations, gene dosage, and gonadal hormones.
The Cav2.1 calcium channels are important modulators of SD (31, 32). The
first evidence that linked Cav2.1 channel mutations to a CSD phenotype was
described in tottering and leaner mice. These naturally occurring mouse mutants
have loss-of-function mutations in their Cav2.1 channels and showed an
increase in SD threshold (33). In a subsequent study, van den Maagdenberg and colleagues (13) demonstrated a reduced
threshold for SD induced by electrical stimulation in R192Q FHM1 mutant mice. The
increased SD frequency and propagation speed following epidural KCl application in R192Q
mutant mice in the present study are in line with these results. Consistent with more
negative opening voltages and a greater increase in the single-channel opening
probability associated with S218L mutation, SD susceptibility was even higher in this
mutant, suggesting incremental modulation of the phenotype by allelic mutations (13, 14, 16). Because excitatory neurotransmitters promote SD
via NMDA receptors (34–38), we speculate that FHM1 mutations facilitate the
initiation and propagation of SDs by increasing presynaptic Ca2+ influx and
subsequent glutamate release. Although augmented neurotransmitter release has been shown
at the neuromuscular junctions of R192Q mutant mice (13, 39), this remains to be tested in
Sex hormones. Our data establish the importance of female gonadal hormones as modulators of
genetically driven enhanced SD susceptibility. Ovarian hormones, rather than sex
chromosome–related developmental differences in synaptic organization and
structure, were implicated in aged female mice and after ovariectomy. These findings
may be clinically relevant, since migraine improves at menopause in two-thirds of
patients (40). The frequency of migraine
attacks decreases after age 50, both in patients with FHM and those with more typical
migraine subtypes (9). Estradiol did partially
restore SD susceptibility when administered chronically to ovariectomized R192Q
mutant mice. Clinically, estrogen reportedly increases cortical excitability in
humans during transcranial magnetic stimulation (41), and high doses increase the incidence of aura during hormone replacement
therapy (42–44). Moreover, higher levels of estrogen are associated with an
increase in seizure frequency in females (45).
Experimentally, seizure thresholds are decreased during peak estrogen levels (46), and amygdala kindling is increased (47). Estrogen augments excitatory glutamatergic
neurotransmission by upregulating NMDA receptor expression, downregulating glutamate
uptake by astrocytes, and increasing the number of dendritic spines, which are
densely populated with NMDA receptors (19–21). A large body of
evidence suggests that estrogen modulates nociceptive processing as well (48, 49),
so that enhanced SD susceptibility is only one mechanism by which estrogen may impact
Clinical and epidemiological data on hormonal modulation of FHM are sparse. In a
population-based study, the female/male ratio was 5:2; males and females did not
differ in age of onset or in symptoms of aura and headache (9). In one case report, a 48-year-old woman with hemiplegic
migraine ceased to experience any further neurological signs during migraine attacks
after ovariectomy (50). Hence, available
evidence supports the notion that female gonadal hormones aggravate the hemiplegic
migraine phenotype as well.
The lack of gonadal hormone modulation of SD in WT mice we observed argues against a
simple additive effect between genetic and hormonal factors and suggests that gonadal
hormones modulate SD susceptibility predominantly in brains made susceptible by gene
mutations. The precise nature of the interactions between gonadal hormones and mutant
Cav2.1 channels remains to be determined. It has been shown, however,
that estrogen upregulates expression of some voltage-gated Ca2+ channels
(e.g., L- and T-type) in hypothalamus and pituitary (51–53). Expression
levels of the α1A subunit of P/Q-type channels show sexual
dimorphism in anterior pituitary (higher in females than in males) and fluctuate
during estrous cycle, suggesting a direct modulation by female hormones (54). Furthermore, Cav2.1 channels
undergo extensive alternative splicing that shows sexual dimorphism (55). Therefore, female hormones may enhance the
impact of genotype on SD phenotype via increased expression or by favoring
alternative splicing patterns that enhance mutant channel activity.
It should be noted that Brennan et al. recently reported that SD thresholds are
reduced by approximately 50% in WT female mice compared with males (56). Using the SD frequency model, we did not
detect sex differences in the WT strain, despite 95% power to detect a 25% difference
between the means in our model (α = 0.05). We also did not detect a
threshold difference using direct epidural cortical electrical stimulation (140
μm tip diameter, 200 μm tip separation, 4.3 kΩ
tip resistance) very similar to that used by Brennan et al. (56) (electrical SD threshold, 413 ± 168 [females] vs.
460 ± 124 [males] μC, n = 11 and 8,
respectively; P = 0.7). At present, we do not have an explanation
for the discrepant results. Nevertheless, consistent with our data, the propagation
speed, SD duration, and number of successful SD inductions did not differ between WT
males and females in their study, and the studies agree that gonadal hormones play an
important part in modulating SD susceptibility.
Motor deficits. Motor deficits (i.e., contralateral hemiplegia with leaning and circling) were
significantly more severe and prolonged in FHM1 mutant mice, as compared with the WT
strain, lasting 20 minutes or more after induction of a single SD. As can be expected
from single-channel kinetics (14), the
deficits were more severe in S218L than in R192Q mutant mice, and often experiments
were terminated by fatal generalized seizures. Delayed electrophysiological recovery
of cortical function did not explain the post-SD deficits (Figure 4). Instead, we believe that the propagation of CSD
into the striatum in FHM1 mutants may have been responsible. The frequency of
striatal SDs (as well as their propagation speed from cortex to striatum) was greater
in the homozygous mutants, in female mutant mice, and in the S218L strain, i.e., the
one with the most severe calcium channel dysfunction (14).
Striatal SDs are associated with contralateral circling and hemiparesis (57–59). CSDs did not propagate into the striatum in WT mice, consistent with the
absence of severe neurological deficits. CSD spreads into the striatum through the
amygdala as SD propagation is impeded in tissues such as white matter with less than
a critical density of neurons (1, 29, 30,
60–62). Consistent with this, direct thalamic and hippocampal
electrophysiological recordings failed to detect concurrent SD propagation into these
structures in either WT or FHM knock-in mouse. In rats, the proportion of CSD
propagating into striatum reportedly varied between 4% and 60% in different studies,
depending on the strain and the use and type of anesthesia, and subcortical
propagation of CSD could be facilitated pharmacologically (e.g., with
pyrrolopyrimidine BW 58271) or physiologically (e.g., in undernourished rats) (30, 59,
60). To our knowledge, this has not been
studied in mice at this level of detail. Our data suggest that FHM1 mutations
facilitate corticostriatal SD propagation. The anesthetic regimen used in our model
may explain the complete lack of corticostriatal SD propagation in WT mice (63, 64).
Post-SD neurological deficits were not only more severe in the mutant strains but
also lasted longer than in WT mice. Indeed, mutants showed one or more episodes of
transient neurological worsening, delaying the neurological recovery after a single
KCl-induced SD. Prolonged deficits and episodes of transient worsening were linked to
recurrent and possibly reentrant SDs in FHM1 mutants. It should also be noted that SD
causes severe and long-lasting tissue hypoperfusion (65) and hypoxia in mice (66). It
remains to be tested whether the hemodynamic and metabolic impact of SD is more
severe in FHM1 mice and whether this contributes to the prolonged post-SD
Seizures. Recurrent SDs observed in FHM1 mutants might have directly caused or predisposed to
delayed seizures in the S218L knock-in. Homozygous female S218L knock-in mice
developed generalized seizures 45–75 minutes after a single SD.
Interestingly, the incidence of premature death at a young age (i.e., <20
weeks, both males and females) was higher in mice of both knock-in strains, in some
cases linked to apparently spontaneous seizures (our unpublished observations),
suggesting that FHM1 mutations predispose to seizures without an experimentally
triggered SD. SD may also render the cortex transiently hyperexcitable. For example,
SD enhances excitatory postsynaptic field potentials as well as the repetition rate
and amplitude of spontaneous rhythmic potentials 20–90 minutes after its
induction; it augments long-term potentiation in human neocortical slices (67, 68).
SD is followed by hyperexcitability in rat neocortex and spinal cord after transient
depression of neuronal activity (69, 70). Last but not least, SD can directly
precipitate seizure-like electrocorticogram activity, as first demonstrated by
Leão (2). Consistent with our
findings, epilepsy is more frequent in FHM patients than in the general population,
with seizures occurring either during FHM attacks (71–74) or interictally
(75–78). Epilepsy has been reported in many patients carrying FHM1
mutations (10, 71, 79–82). For example, 2 patients harboring the S218L
mutation have developed seizures at the onset of severe attacks associated with coma
In summary, data showing enhanced SD susceptibility and prolonged neurological
deficits after SD in genetic mouse models of migraine strengthen the link between SD
and the migraine aura. Furthermore, they provide mechanistic insight by implicating
recurrent SDs and facilitated propagation of SD into subcortical tissues. The
modulation of SD susceptibility, neurological deficits, and subcortical propagation
by specific FHM1 mutations (i.e., R192Q vs. S218L mutation), genotype (i.e.,
heterozygous vs. homozygous), and gonadal hormones underscores the complex
synergistic interactions between genetic and hormonal factors determining migraine
Experimental groups. Experimental groups are summarized in Table 1.
A total of 337 mice were used in this study. Male and female FHM1 mutant mice,
homozygous or heterozygous for R192Q or S218L mutation in the mouse
Cacna1a gene (encoding the α1A pore-forming
subunit of Cav2.1 channels), were compared with WT littermates. In
addition, R192Q mutants were backcrossed onto C57BL6/J mice (Charles River
Laboratories) for more than 8 generations. None of the measured endpoints differed
between WT littermates of R192Q mutant and C57BL6/J control mice (data not shown).
Therefore, data from WT littermates of R192Q mutants and C57BL6/J control mice were
pooled for the analysis. The S218L mutants were compared with their littermates only.
Preparation of targeting construct and generation of FHM1 mutant mice. Transgenic knock-in Cav2.1-α1A migraine mouse
models were generated by a gene targeting approach in which the endogenous
Cacna1a gene was modified. In the R192Q mice (13), we modified the CGG triplet (arginine) of
codon 192 to CAG (glutamine) using in vitro mutagenesis to introduce the human FHM1
mutation R192Q (12). The targeting vector that
was used for generating the S218L mice contained the mutant TTA (leucine) instead of
the original TCA (serine) triplet of codon 218; thus creating the S218L mutation that
had previously been found in patients (15).
This targeting vector also contained a PGK-driven neomycin-resistance cassette that
was flanked by loxP sites in the endogenous HindIII
site that is located 537 nucleotides upstream of exon 5. Chimeras were obtained by
injecting correctly targeted E14 ES cells into C57BL/6J blastocysts according to
standard procedures in order to generate a transgenic line of mice. In mice that were
used for the experiments, the cassette was deleted by crossing these transgenic mice
with mice of the EIIA-Cre deleter strain (84),
which express Cre recombinase driven by the EIIA early promoter. For both the R192Q
and S218L mice, heterozygous mice (>96% C57BL/6J background) were subsequently
interbred to provide litters containing all 3 possible genotypes that were used for
the experiments. Litters were genotyped after weaning by PCRs specific for the
respective transgenic line, essentially as described previously (13). All experiments were carried out with the
investigator blinded for genotype, and confirmatory genotyping was done after the
experiment. All animal experiments were approved by the Massachusetts General
Hospital Subcommittee on Research Animal Care and performed in accordance with the
NIH Guide for the care and use of laboratory animals (NIH
publication no. 85-23. Revised 1985).
General surgical and electrophysiological procedures. Mice were housed under diurnal lighting conditions and allowed food and tap water ad
libitum. To assess SD susceptibility under full systemic physiological monitoring,
femoral artery was catheterized for blood sampling and measurement of mean arterial
pressure and trachea intubated for mechanical ventilation (SAR-830; CWE), under
isoflurane anesthesia (2.5% induction, 1% maintenance, in 70% N2O/30%
O2). Arterial blood gases and pH were measured once every 20 minutes
during each experiment in 25 μl samples (Corning 178 blood gas/pH
analyzer; Ciba Corning Diagnostic Corp.) and maintained within normal limits by
adjusting ventilation parameters (Table 1).
Mice were then placed in a stereotaxic frame (David Kopf Instruments), and 3 burr
holes were drilled under saline cooling at the following coordinates on both sides
(mm from bregma): 3.5 mm posterior, 2 mm lateral (2 mm diameter for KCl application
onto occipital cortex); 1.5 mm posterior, 2 mm lateral (1 mm diameter, recording site
1); 0.5 mm anterior, 2 mm lateral (1 mm diameter, recording site 2). The dura was
kept intact to minimize trauma. Two glass capillary microelectrodes were placed to
record extracellular steady (DC) potential and electrocorticogram (ECoG) at a depth
of 300 μm. For striatal recordings, the electrode at recording site 2 was
lowered to a depth of 3 mm into the striatum. A third electrode was simultaneously
inserted at 2 mm posterior, 1.2 mm lateral from bregma, at depths of 3 and 1.2 mm
from the pial surface for thalamic and hippocampal recordings, respectively.
Recording sites were later confirmed by tracking the electrode placement
(Supplemental Figure 2). An Ag/AgCl reference electrode was placed subcutaneously in
the neck. Recording sites were covered with mineral oil after electrode placement to
prevent cortical drying. After surgical preparation, the occipital cortex was allowed
to recover for 20 minutes under saline irrigation. A cotton ball (2 mm diameter)
soaked with 300 mM KCl was placed on the dura and replaced every 15 minutes to
maintain steady KCl concentration during stimulation. In order to test whether
occipital epidural KCl application causes SD in the striatum via direct diffusion, in
a subgroup of FHM1 mutant mice we evoked SD by pinprick on the occipital cortex and
observed reproducible propagation of SD into striatum with the same latency as after
topical KCl application (n = 3 female R192Q mutant mice; data not
shown). All data were continuously recorded using a data acquisition system for
off-line analysis (PowerLab; ADInstruments).
The frequency of SDs evoked by topical KCl application and their propagation speed
were determined as previously described with minor modifications (4). KCl-evoked CSDs were detected based on the
characteristic slow DC potential shift and ECoG suppression. The frequency of evoked
CSDs was determined over 30 minutes on each hemisphere. CSD frequencies obtained from
the right and left hemispheres did not statistically differ in any experimental group
and were averaged to calculate the CSD frequency in each mouse. Propagation speed was
calculated from the distance between the 2 recording electrodes divided by the
latency between the first CSDs recorded at these sites. The DC shift amplitude and
duration at half-maximal amplitude were averaged for all CSDs in each experiment.
Epidural application of NaCl (300 mM) did not trigger SD or cause neurological
deficits in sham-operated knock-in controls (n = 2 homozygous female
R192Q mutant mice; data not shown). In subgroups of WT and FHM1 mutant mice,
histopathological assessment of the KCl application site after the experimental
recordings did not reveal any evidence of cortical injury in H&E-stained
frozen sections (data not shown).
Ovariectomy and estrogen replacement. The impact of diminished gonadal hormone production on SD susceptibility was studied
using ovariectomized (3–4 months old) or senescent (13 months old, after
cessation of estrous cycle) homozygous R192Q mutant mice. Because the sex effect on
SD phenotype appeared in both heterozygous and homozygous mutants (Figure 1), further experiments exploring post-SD
neurological deficits (see below) were performed on homozygous mutants only. Ovaries
were exteriorized, ligated, and removed via bilateral dorsal approach in young adult
mice (3–4 months old) under isoflurane anesthesia. In addition, subgroups
of ovariectomized mice were chronically treated with estrogen via subcutaneous
implantation (dorsal scapular) of 21-day slow-release pellets (Innovative Research of
America) containing 17β-estradiol 3-benzoate to achieve constant plasma
estrogen levels corresponding to the proestrus stage of cycle (0.025 mg/pellet) or
higher-than-normal estradiol concentration (0.075 mg/pellet) (85–90). SD
susceptibility was tested 3 weeks after ovariectomy with or without estrogen
replacement (i.e., empty pellet implantation). The C57BL/6J background of FHM1 mutant
mice has a gonadal hormone profile of aging very similar to that observed in
menopausal women: prolonged cycles with delayed preovulatory rise of estrogen
progress to acyclicity, lower estrogen levels, and hypergonadotrophic hypogonadism
(91, 92); decreased nuclear estrogen receptors and a nuclear translocation defect
of estrogen-receptor complex have also been described as in humans (93–95).
Neurological testing. In order to minimize the confounding effect of invasive surgical procedures on
neurological testing, arterial and tracheal catheterizations were not performed, and
freely breathing mice were anesthetized via a face mask during the SD induction
procedure. Neurological deficits were assessed either after 1 SD or after 9 SDs
induced over 1 hour. In the 1-SD group, mice were briefly anesthetized, and KCl (300
mM) was topically applied (typically for less than 1 minute) on the parietooccipital
cortex until an SD was recorded in the frontal cortex as described above, followed by
extensive saline wash of cortical surface. In the 9-SD group, KCl (300 mM) was
briefly applied in the same manner approximately every 7.5 minutes, and SD induction
confirmed at the frontal recording site. Motor deficits were assessed at predefined
time points after induction of the last SD in a blinded fashion. Mice fully awakened
from anesthesia usually within 3 minutes in the 1-SD group; therefore, neurological
assessments were started 5 minutes after SD induction and carried out every 5 minutes
until full reversal of deficits. Because full awakening was delayed in the 9-SD group
for up to 15–20 minutes in all groups, first neurological assessment was
carried out 30 minutes after the last SD and then repeated at 45, 80, and 120
minutes. The severity of deficits and high mortality after a single SD in S218L
mutant mice precluded testing of the 9-SD paradigm in this strain. There was no
difference among WT and FHM1 mutant strains in the time required for full recovery of
neurological function after 10 minutes of isoflurane anesthesia (data not shown).
We relied exclusively upon motor performance, since preliminary studies of sensory
deficits and neglect (e.g., corner test) did not reliably detect deficits in this
model (data not shown). Motor deficits were assessed using 2 independent tests. The
5-point neurological scale is most commonly used to quantify unilateral motor
deficits after stroke. The deficits are scored as: 0 (no neurological deficit:
normal), 1 (mild neurological deficit: failure to extend forepaw fully), 2 (moderate
neurological deficit: circling), 3 (severe neurological deficit: falling to one
side), 4 (very severe neurological deficit: no spontaneous walking, depressed level
of consciousness), as previously described (96). The wire grip test is used to assess coordination and fine movement of
the digits based on the ability of the mouse to remain on the wire and successfully
climb down the pole and scored as: 0, unable to remain on wire more than 30 seconds;
1, holds on wire more than 30 seconds, but not with both sets of paws on wire; 2,
holds on to the wire with all paws but not the tail; 3, uses the tail along with all
paws but does not move on wire; 4, moves along the wire on all 4 paws plus tail; 5,
moves along the wire on all 4 paws plus tail and ambulates down one of the posts used
to support the wire. Besides the wire grip score, we also recorded the latency to
fall off the wire; if the mouse stayed on the wire for more than 60 seconds, or
climbed down the pole successfully, it was scored as 60 seconds (97).
Somatosensory evoked potentials. Two cranial windows (1 mm diameter) were drilled under saline cooling over the right
whisker barrel cortex (posterior 1.3 mm, lateral 3.7 mm from bregma) and occipital
cortex (posterior 4 mm, lateral 2 mm from bregma). The dura was kept intact. A glass
micropipette electrode filled with 150 mM NaCl was inserted to a depth of 400
μm. Somatosensory evoked potentials as well as the extracellular steady
(DC) potential were recorded using a differential amplifier (EX-1; Dagan Corp.) and
stored using a data acquisition system for off-line analysis (PowerLab 200;
ADInstruments). An Ag/AgCl reference electrode was also placed subcutaneously in the
neck, and the cortex was covered with a thin layer of mineral oil to prevent drying.
Somatosensory potentials in the whisker barrel cortex were evoked by electrical
stimulation of the entire whisker pad (single square pulse, 0.2 ms duration, 700
μA, 0.1 Hz; S48, Grass Technologies; and A395 Linear Stimulus
Isolator/Constant Current Unit, WPI) via 2 needle electrodes placed in the
contralateral whisker pad (5 mm electrode separation). In preliminary experiments, we
determined that nitrous oxide in inhalation gas potently suppressed somatosensory
evoked potentials. Therefore, in these experiments, we replaced nitrous oxide with
Whisker pad stimulation typically evoked a monophasic negative field potential at
this recording depth (Figure 4). After acquiring
50 such evoked potentials at baseline, a CSD was triggered using brief occipital
epidural KCl application and confirmed by the characteristic DC potential shift at
the recording site in whisker barrel cortex. In order to avoid multiple CSDs, the KCl
application site was immediately washed with saline upon detection of the SD at the
recording site. Experiments with multiple SDs were excluded from analysis. Both the
amplitude of negative peak and the area under the field potential curve were measured
before, during, and after SD and expressed as percent of baseline.
Statistics. Data were analyzed using SPSS software package (version 11.0). The impact of
independent variables allelic mutations (R192Q vs. S218L), allele dosage (WT,
heterozygous, homozygous), and sex (male vs. female) on the dependent variables
cortical and striatal SD frequency and propagation speed were tested using 3-way
ANOVA (Figures 1 and 5). The impact of independent variables R192Q mutation (WT vs.
R192Q) and gonadal status (normal, ovariectomized, and senescent) on the dependent
variables CSD frequency and propagation speed were tested using 2-way ANOVA (Figure
2). The time course of neurological deficits
and recovery of somatosensory evoked potentials after SD were tested among
experimental groups using 2-way ANOVA for repeated measures (Figures 3 and 4).
Electrophysiological measures of CSD (Table 1), systemic physiological data (Table 1), and the impact of estrogen replacement in ovariectomized FHM1 mutant mice
(see Results) were compared among experimental groups using 1-way ANOVA.
Corticostriatal propagation latencies were compared using 3-way ANOVA (Table 2). In addition, using pooled data from all mice
and a general linear model of covariance analysis (ANACOVA), we tested for an effect
of the independent variables mutation, genotype, and sex (fixed factors) on the
dependent variables cortical and striatal SD frequency and propagation speed. Data
are presented as mean ± standard deviation. P <
0.05 was considered statistically significant.
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This work was supported by the Deutsche Forschungsgemeinschaft (Ha5085/1-1 to K.
Eikermann-Haerter), National Institute of Neurological Disorders and Stroke
(1R01NS061505 to C. Ayata; 2P01NS35611 to M.A. Moskowitz), Netherlands Organization for
Scientific Research (903-52-291 and Vici 918.56.602 to M.D. Ferrari), EU
“EUROHEAD” grant (LSHM-CT-2004-504837 to M.D. Ferrari, A.M.J.M.
van den Maagdenberg), and the Centre for Medical Systems Biology (CMSB) in the framework
of the Netherlands Genomics Initiative (NGI). We would like to thank Elkan Halpern and
Tobias Kurth for statistical guidance, Lynda Banzi for assistance with videos, Mike
Whalen and Zerong You for assistance with neurological deficit assessment, and Jianhua
Qiu for histological imaging.
Conflict of interest: The authors have declared that no conflict of
Nonstandard abbreviations used: CSD, cortical SD; FHM, familial
hemiplegic migraine; SD, spreading depression.
Citation for this article:J. Clin. Invest.119:99–109 (2009). doi:10.1172/JCI36059
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