neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Transgenic mice that overexpress the G93A mutation of the
human Cu-Zn superoxide dismutase 1 gene (hSOD1G93Amice) are a commonly used animal model of ALS. Whole-cell patch-clamp
increased persistent Na?current (PCNa), and enhanced frequency of spontaneous excitatory and inhibitory transmission, compared
2–3 months before motoneuron degeneration and clinical symptoms appear in these mice. Changes in neuronal activity were not
eling, and behavioral assays revealed transient neonatal neuromotor deficits compared with controls. These findings underscore the
Amyotrophic lateral sclerosis (ALS) is a fatal paralytic disorder
caused by adult degeneration of cranial and spinal motoneurons
(Mulder, 1982). Average survival from symptom onset is 3–5
years, and treatment with riluzole only prolongs patient survival
by a few months (Cleveland and Rothstein, 2001). Approxi-
dominant mutations in the Cu-Zn superoxide dismutase 1
(SOD1) gene (Rosen et al., 1993). As in FALS patients, mice
progressive degeneration of motoneurons in adulthood (Gurney
mutant gene and the proximal causes of motoneuron death in
these intensively studied mouse lines remains obscure. Recent
studies have provided important insights as to which cell types
contribute to ALS pathology. Although mutant SOD1 causes in-
trinsic damage to motoneurons, non-neuronal cells may also
al., 2003; Boille ´e et al., 2006; Di Giorgio et al., 2007; Nagai et al.,
2007). In vitro experiments show that conditioned media from
wild-type (WT) motoneurons (Nagai et al., 2007). However, it is
still unclear how mutant hSOD1 in neuronal and non-neuronal
cells causes motoneuronal pathology and death.
Studies with mutant hSOD1 transgenic mice in vivo and in
Power for advice on hypoglossal motoneuron electrophysiological experiments and J. Durand for advice on the
packaged by R.L.N. Behavioral testing was performed and analyzed by M.H. and A.C. B.v.Z., R.H.B., M.C.-P., and
Correspondence should be addressed to Mark C. Bellingham, School of Biomedical Sciences, University of
10864 • TheJournalofNeuroscience,October22,2008 • 28(43):10864–10874
toneurons, including excitotoxicity, disturbed Ca2?homeosta-
sis, mitochondrial dysfunction, SOD1 aggregation, cytoskeletal
disruption, activation of cell death signals, and oxidative stress
(Brown and Robberecht, 2001; Cleveland and Rothstein, 2001;
Pasinelli and Brown, 2006). Nonetheless, the origin(s) and
pathogenesis of motoneuron death in ALS remain largely un-
known, because of the difficulty in distinguishing between pri-
mary target(s) of mutant SOD1 and secondary effects and com-
pensatory mechanisms. This is particularly true for genetically
induced diseases, in which effects of early expression of the aber-
rant gene can cause compensatory activity-dependent events in
the developing CNS, masking overt symptoms until compensa-
In hSOD1G93Amice, several pathogenic events in motoneu-
rons, including mitochondrial dysfunction [as early as postnatal
day 14 (P14)] and caspase-1 activation (P70), occur before clin-
al., 1994; Bendotti et al., 2001). To analyze functional changes in
neuronal activity occurring during early development, we per-
formed whole-cell patch-clamp recordings from hypoglossal
motoneurons (HMs) in acutely prepared brainstem slices from
sistent Na?current (PCNa), hyperexcitability, and spontaneous
synaptic transmission in these motoneurons. Similar functional
neurons (P10–P12), in which a precise timetable of synaptic de-
velopment has been established in wild-type mice and in which
we observed clearly precocial synaptic changes in hSOD1G93A
mice. We also observed structural alterations in motoneuron
dendritic architecture and locomotor behavior changes, consis-
PCNaand hyperexcitability in motoneurons suggests that these
changes could contribute to later motoneuron death in the adult
Animals. All mice handlings were in accordance with the United States
National Institute of Health guidelines and were approved by the Insti-
tutional Animal Care and Use Committees of Massachusetts Institute of
Technology and Massachusetts General Hospital. In this study, hemizy-
gous transgenic B6SJL mice carrying a high copy number of mutant
human SOD1 (hSOD1G93A) and WT human SOD1 (hSOD1WT) were
originally obtained from Jackson Laboratories. Nontransgenic litter-
mates (mSOD1WT) and hSOD1WTmice were used as controls. Trans-
genic mice were identified by PCR as described earlier (Rosen et al.,
1993). Genotyping was performed after the electrophysiological experi-
ments had been performed to allow the experimenter to be “blind” to
animal genotype. The hSOD1G93Amice, but not the mSOD1WTor
hSOD1WTmice, develop initial signs of neuromuscular deficits (tremor
of the legs and loss of extension reflex of the hindpaws) at ?10 weeks of
age. At 18 weeks, they show marked locomotor impairment, with paral-
ysis and muscular atrophy of the hind limbs. These animals die of respi-
ratory failure at 20–21 weeks of age.
Slice preparations. Acute brainstem (P4–P10) and midbrain (P10–
P12) slices were prepared as described previously (Bellingham and
Berger, 1996; O’Brien and Berger, 1999; Aamodt et al., 2000; Shi et al.,
2000; Townsend et al., 2003; Ireland et al., 2004). Animals were anesthe-
tized with isoflurane and decapitated. The brainstem or the midbrain
were quickly isolated and cut in 300- to 350-?m-thick transverse or
tion containing the following (in mM): 130 NaCl, 26 NaHCO3, 1.25
NaH2PO4, 3 KCl, 10 glucose, 1 CaCl2, and 5 MgCl2, pH 7.35. Slices were
at least another 0.5 h at room temperature (RT) in the same solution,
except with 2 mM CaCl2and 2 mM MgCl2. Solutions were continuously
bubbled with a 95% O2–5% CO2gas mixture.
Electrophysiology. Whole-cell recordings were made from HMs in
brainstem slices (nXII) (Bellingham and Berger, 1996; O’Brien and
Berger, 1999; Ireland et al., 2004) and SC interneurons in the stratum
et al., 2000; Townsend et al., 2003). Recording artificial CSF (ACSF)
3 KCl, 10 glucose, 2 CaCl2, and 1–4 MgCl2, bubbled with 95% O2/5%
CO2, pH 7.35. For recording action potential (AP) firing and voltage-
dependent currents, a K gluconate-based internal solution was used,
synaptic currents, a Cs gluconate-based internal solution was used, con-
taining the following (in mM): 122.5 Cs gluconate, 17.5 CsCl, 8 NaCl, 10
HEPES, 0.2 EGTA, 2 MgATP, and 0.3 GTP-Na. After formation of a
high-resistance seal (?1G?) and break-in, only neurons with series re-
current signals were recorded with an Axopatch 200B amplifier (Molec-
ular Devices). Pipette and whole-cell capacitance and series resistance
were compensated using amplifier circuitry. Signals were low pass fil-
tered (1–10 kHz) and digitized (5–40 kHz) on a PC using pClamp soft-
ware, and measured and analyzed with Clampfit 8.0–10, MiniAnalysis
(Synaptosoft), Excel (Microsoft), and Prism 4 (Graphpad).
Recording of AP firing and voltage-dependent currents were per-
formed with complete blockage of ionotropic synaptic transmission by
(10 ?M DNQX), NMDA receptors (NMDARs) (50 ?M D-APV), glycine
receptors (GlyRs) (2 ?M strychnine HCl), and GABAAreceptors
(GABAARs) [5 ?M bicuculline methiodide (BMI)]. To record synaptic
GlyR, different mixtures of these antagonists were applied to the bath
Townsend et al., 2003) and for HMs (Bellingham and Berger, 1996;
were isolated by the addition of D-APV, strychnine, and bicuculline. The
remaining synaptic currents were AMPAR mediated, because they were
completely abolished after recordings by bath application of DNQX. In
some experiments, quantal AMPAR-mediated currents were isolated by
additional bath application of the sodium channel blocker TTX (0.5–1
?M) with or without bath application of CdCl2(100 ?M). Spontaneous
NMDAR-mediated EPSCs were isolated in Mg2?-free ACSF by the ad-
dition of DNQX, strychnine, and BMI. The remaining synaptic currents
were NMDAR mediated, because they were completely abolished subse-
mediated IPSCs were isolated by the addition of D-APV and CNQX. In
SC interneurons, these IPSCs were mediated only by GABAARs, because
they were completely abolished by subsequent bath application of BMI
(Aamodt et al., 2000; Shi et al., 2000). In HMs, the IPSCs were mediated
by a mixture of GlyRs and GABAARs, because addition of both strych-
ings, neurons were held at a membrane potential of ?70 mV, except for
recorded at ?50 mV for comparison to previous SC interneuron data
(Aamodt et al., 2000; Shi et al., 2000).
continuous recording, chosen randomly throughout the recording pe-
riod for each neuron. Baseline noise ranged from 2.5 to 5.0 pA peak to
peak, and synaptic events with an amplitude ?2 times 1/2 peak-to-peak
noise were analyzed. Synaptic currents were characterized by the follow-
ing parameters: peak amplitude, interval between sequential currents,
rise time (from 10 to 90% of peak amplitude), and decay time. Decay
peak amplitude with a single exponential and is given in milliseconds
vanZundertetal.•NeonatalHyperexcitabilityinMouseALSModelJ.Neurosci.,October22,2008 • 28(43):10864–10874 • 10865
the fitted exponential. The mean for each parameter for all synaptic
sis 5.1 and Prism 4.
HM labeling. Construction and packaging of HSV amplicon vector
p1003 containing the green fluorescent protein (GFP) driven by a CMV
promoter has been described previously (Neve et al., 2005; Olson et al.,
2005). To retrogradely label HMs in the ipsilateral hypoglossal nucleus
(nXII), P1 animals were deeply anesthetized by hypothermia and 1–2 ?l
of HSV-GFP p1003 (2 ? 108/ml) was slowly pressure-injected into the
tip of the tongue muscle with a borosilicate glass electrode pipette (?50
until they recovered spontaneous movement and allowed to survive till
P6 before being killed. For immunohistochemistry, mice were deeply
anesthetized with pentobarbital, and intracardially perfused with 4%
fixed overnight in the same fixative, and maintained in a 30% sucrose-
the infected HMs, 250 ?m sequential vibratome sections through the
entire brainstem were cut, and sections were permeabilized in 0.5% Tri-
ton X-100/PBS for 2 h at RT and then blocked with 10% normal horse
antibody coupled to Alexa-488 (Invitrogen) overnight at 4°C in 0.025%
Triton X-100/PBS. After washing and mounting, the sections were ana-
lyzed using a Nikon PCM 2000 confocal microscope equipped with an
argon laser and appropriate performance filters for detection of GFP.
Behavioral testing. Six sensorimotor responses appearing from P1 to
P12 were measured daily in all male and female mice as described previ-
ously (Amendola et al., 2004). When the appropriate responses were
observed, the pup was given a score of 1 for the corresponding test. The
following tests were performed: (1) righting: the pup was placed on its
the edge) and was tested to turn and crawl away from the cliff. (3) Fore-
paw and (4) hindpaw grasping: the inside of one paw of the pup was
object was tested. (5) Forelimb placing: the dorsum of one paw was
it on the object was tested. (6) Vibrissae placing: the pup was suspended
with the extended forelimb when the vibrissae touched a pencil.
significant differences were determined using Student’s unpaired t test
(electrophysiology and structural changes) or ?2test (animal behavioral
studies) except where noted. For all statistical tests, values of *p ? 0.05,
**p ? 0.01, and ***p ? 0.001 were considered statistically significant.
As in FALS patients, hSOD1G93Amice display a progressive de-
generation of motoneurons in adulthood (beginning at 2.5–3
months after birth) (Gurney et al., 1994). This restricted cell
death of adult motoneurons cannot be explained by the spatio-
temporal expression of mutant SOD1, because hSOD1G93Ain
all cells of the CNS throughout life (Gurney et al., 1994). To
analyze whether normal brain function is disturbed during early
development, we performed whole-cell patch-clamp recordings
from HMs in acutely prepared brainstem slices from P4–P10
hSOD1G93Aand mSOD1WTmice. We first analyzed whether
HMs in brain slices display increased intrinsic excitability at this
early stage of development, as occurs in dissociated cultured spi-
nal cord neurons derived from embryonic hSOD1G93Amice
(Kuo et al., 2004). To analyze intrinsic excitability, we recorded
AP firing in response to depolarizing current steps (Fig. 1A). We
found that hSOD1G93AHMs fired at significantly higher rates
than HMs in brainstem slices from mSOD1WTmice (Fig. 1A,B);
mean firing rates with current injections of 80 and 120 pA were
22.9 ? 1.9 and 29.7 ? 2.1 Hz in 12 mSOD1WTHMs, and 32.7 ?
2.4 and 40.0 ? 3.7 Hz in 6 hSODG93AHMs (repeated-measures
ANOVA, Bonferroni posttest). In addition, we also analyzed fir-
ing rates of HMs from hSOD1WTmice. We found that the firing
120 pA; data not shown) were similar to mSOD1WTHMs and
significantly lower than those of hSOD1G93AHMs (repeated-
measures ANOVA, Bonferroni posttest). This finding supports
the idea that motoneuron hyperexcitability was specifically at-
brainstem slices from early postnatal hSODG93A-overexpressing
mice show intrinsic hyperexcitability, 2–3 months before cell
death and clinical symptoms appear.
To investigate the basis for this hyperexcitability, we analyzed
several cell properties and AP characteristics of mSOD1WTand
hSOD1G93AHMs. Although no differences in AP firing thresh-
old, AP duration, afterhyperpolarization amplitude, resting
membrane potential, or input resistance were found between
mSOD1WTand hSOD1G93AHMs, the AP amplitude of
hSOD1G93AHMs was significantly increased compared with
mSOD1WTmotoneurons (Table 1). As inhibition of 60% of
mice. HMs in acutely prepared brainstem slices of mSOD1WTand hSOD1G93Amice were re-
Intrinsic excitability is increased in HMs from presymptomatic hSOD1G93AALS
10866 • J.Neurosci.,October22,2008 • 28(43):10864–10874vanZundertetal.•NeonatalHyperexcitabilityinMouseALSModel
nel density may contribute to hyperexcitability in developing
hSOD1G93AHMs. Accordingly, we measured the maximum rate
of rise of individual APs, because this measurement is more di-
icantly increased in HMs from hSODG93Amice (Table 1).
PCNa, capable of amplifying a neuron’s response to excitatory
prolonged depolarization (Kuo et al., 2005). To test whether a
PCNais present in early postnatal HMs and contributes to regu-
lation of HM repetitive firing, we bath applied riluzole (20 ?M).
Riluzole inhibits PCNabut has relatively small inhibitory effects
on the fast inactivating Na?current, which generates APs (Ur-
bani and Belluzzi, 2000). Riluzole did not prevent AP generation
at the current step onset but significantly reduced repetitive AP
firing in response to current injection in mSOD1WTHMs (Fig.
2A), demonstrating that PCNawas present in neonatal HMs.
Next, we directly recorded isolated PCNato determine
whether PCNa is increased in hSOD1G93AHMs. Voltage-
dependent inward currents were evoked by slow voltage ramps
with bath-applied CdCl2(100 ?M) to block voltage-gated Ca2?
channels (Carlin et al., 2000; Powers and Binder, 2003). In both
mSOD1WTand hSOD1G93AHMs, an inward current was pro-
at ?30 to ?10 mV (Fig. 2B1–B4, gray line). This inward current
was a PCNa, because it was abolished by either riluzole (20 ?M)
a highly specific Na?channel antagonist that blocks both PCNa
and AP generation (Goldin, 1999). After normalizing whole-cell
current for cell membrane area (determined by measurement of
whole-cell capacitance), the mean peak amplitude of the PCNa
icantly increased ( p ? 0.05, unpaired t test), approximately
alterations in the voltage dependence of PCNa(Fig. 2C). To-
gether, these results strongly suggest that increased PCNais a key
mechanism causing intrinsic hyperexcitability in early postnatal
HMs from hSOD1G93Amice.
We reasoned that if generation and repetitive firing of APs is
an essential feature of most neurons and expression of mutant
SOD1 is ubiquitous in CNS neurons, intrinsic hyperexcitability
might not be unique to motoneurons in hSOD1G93Amice. Hy-
perexcitability might therefore also occur in interneurons, such
as in SC interneurons that control eye movements, a function
that remains relatively normal in human ALS patients until the
final stages (Kaminski et al., 2002). In addition, SC interneurons
undergo well characterized and precisely timed developmental
changes in synaptic transmission. At the end of the second post-
natal week (P13–P14), when visual activity increases with retinal
maturation and eye-opening, frequency of spontaneous synaptic
increases, whereas NMDAR currents (sNMDAs) decay faster be-
cause of a change in the composition from NR2B-rich to NR2A-
rich receptors (Shi et al., 1997, 2000; Aamodt et al., 2000; Colon-
nese et al., 2003; Lu and Constantine-Paton, 2004; Townsend et
al., 2004; van Zundert et al., 2004). The dependence of these
synaptic changes on neural activity led us to predict that hyper-
aptic transmission in hSOD1G93Amice.
dicted, hSOD1G93ASC interneurons in acutely prepared mid-
brain slices from hSOD1G93AP10–P12 mice also fired at higher
frequencies (30.9 ? 4.3 Hz at 120 pA, n ? 7) than SC interneu-
at 120 pA, n ? 8; p ? 0.05, repeated-measures ANOVA, Bonfer-
traces in a mSOD1WTHM without (A1) and with (A2) riluzole (20 ?M). Note that riluzole is
unable to prevent AP generation at the current step onset but abolishes repetitive AP firing
mean PCNacurrent normalized to cell capacitance (current density) plotted against voltage
ramp membrane potential (MP) for mSOD1WTHMs (n ? 9) and hSOD1G93AHMs (n ? 12),
vanZundertetal.•NeonatalHyperexcitabilityinMouseALSModel J.Neurosci.,October22,2008 • 28(43):10864–10874 • 10867
amplitude in hSOD1G93ASC interneurons, no differences in
other AP characteristics or cell properties were found between
mSOD1WTand hSOD1G93ASC interneurons, including maxi-
ronal loss in spinal cord and cortex of human ALS patients
(Maekawa et al., 2004; Stephens et al., 2006) and in transgenic
mice overexpressing mutant hSOD1 (Morrison et al., 1998), this
suggests that early hyperexcitability in interneurons may also
contribute to interneuronal loss at later stages in ALS.
On the basis of our finding that intrinsic excitability is increased
excitable neuronal networks in early postnatal hSOD1G93Amice
would generate increases in activity-dependent release of excita-
tory and inhibitory neurotransmitters. Enhanced activity-
aptic neurons as an increase in spontaneous EPSCs and IPSCs
(Bellingham and Berger, 1996; O’Brien and Berger, 1999). In SC
interneurons, synaptic inputs are from other local excitatory or
inhibitory SC interneurons (Aamodt et al., 2000; Shi et al., 2000;
Townsend et al., 2003), whereas synaptic inputs to HMs are
largely from excitatory and inhibitory premotoneurons in the
reticular formation lateral to the hypoglossal motor nucleus
(Dobbins and Feldman, 1995; Bellingham and Berger, 1996;
O’Brien and Berger, 1999).
As predicted, the mean interevent interval of sAMPAs
n ? 12; p ? 0.0067) and sGABAs (mSOD1WT? 2.50 ? 0.45 s,
n ? 5; hSOD1G93A? 1.07 ? 0.21 s, n ? 11; p ? 0.036) was
significantly shorter in P10–P12 hSOD1G93ASC interneurons
compared with mSOD1WTSC interneurons (Fig. 4A, traces; Fig.
4C for distribution of means). Mean amplitude of sAMPA
(mSOD1WT? 13.4 ? 0.91 pA; hSOD1G93A? 15.6 ? 1.55 pA;
p ? 0.21) or sGABA (mSOD1WT? 36.6 ? 3.78 pA; hSOD1G93A
ent in SC interneurons from hSOD1G93Amice (Fig. 4B for dis-
utable toincreased activity-dependent
the presence of TTX (500 nM) demonstrated that the mean am-
plitude (mSOD1WT? 14.3 ? 1.73 pA, n ? 6; hSOD1G93A?
14.4 ? 1.15 pA, n ? 7; p ? 0.92) and interevent interval
(mSOD1WT? 2.96 ? 1.38 s; hSOD1G93A? 2.37 ? 0.67 s; p ?
0.70) of mAMPA events in hSOD1G93ASC interneurons were
similar to those in mSOD1WTSC interneurons (Fig. 4A, traces;
EPSC activity and GABAergic IPSC activity normally increase
these findings also suggested that the enhanced spontaneous
transmission in SC interneuron networks was attributable to an
acceleration of normal synaptic development because of in-
To further investigate whether accelerated synaptic develop-
ment occurred in hSOD1G93Amice, we also recorded sNMDAs
interneurons (Shi et al., 1997, 2000; Colonnese et al., 2003;
Townsend et al., 2003, 2004; Lu and Constantine-Paton, 2004).
0.30 pA, n ? 6; hSOD1G93A? 10.13 ? 0.51 pA, n ? 5; p ? 0.37)
or interevent interval (mSOD1WT? 6.61 ? 3.13 s; hSOD1G93A
? 2.24 ? 0.20 s; p ? 0.24) were not altered in hSOD1G93ASC
interneurons (Fig. 4A for traces; Fig. 4B,C for distributions of
means), suggesting that hSOD1G93ASC interneurons may have
hSOD1G93AALS mice. SC interneurons of acutely prepared midbrain slices of mSOD1WTand
Intrinsic excitability is increased in SC interneurons from presymptomatic
(P10–P12). AP threshold is relative to resting membrane for each SC recorded; AP and afterhyperpolarization
10868 • J.Neurosci.,October22,2008 • 28(43):10864–10874vanZundertetal.•NeonatalHyperexcitabilityinMouseALSModel
taneous EPSCs decrease with age in SC interneurons (Shi et al.,
1997). Analyses of sNMDA current kinetics also showed that the
10–90% rise time (mSOD1WT? 2.25 ? 0.29 ms; hSOD1G93A?
1.74 ? 0.15 ms; p ? 0.18) was not altered, but that decay time
(mSOD1WT? 21.33 ? 3.73 ms; hSOD1G93A? 10.2 ? 2.03 ms;
p ? 0.036) of sNMDA currents was significantly faster in
hSOD1G93ASC interneurons (Fig. 4C,D for distributions of
rons is well characterized and can be induced by changes in the
subunit composition, in which slow-decaying NR2B-containing
receptors are replaced by fast-decaying NR2A-containing recep-
tors (Shi et al., 2000; Townsend et al., 2003; Lu and Constantine-
Paton, 2004). To test whether sNMDA currents are no longer
predominantly mediated by NR2B-containing NMDA receptors
in P10–P11 hSOD1G93ASC interneurons, we used ifenprodil,
which preferentially antagonizes NR2B-
containing NMDA receptors (Williams,
P10–P11 hSOD1G93Amice were insensi-
tive to 5 ?M ifenprodil (control/ifen-
a developmental NR2B-to-NR2A subunit
We then recorded spontaneous and/or
quantal synaptic currents mediated by
AMPA, NMDA, and inhibitory GABAA
and glycine receptors (sIPSCs) in early
postnatal mSOD1WTand hSOD1G93A
HMs. As for SC interneurons, we found
that the mean interevent intervals of
hSOD1G93A? 0.56 ? 0.05 s, n ? 5; p ?
0.009) and sIPSC (mSOD1WT? 2.53 ?
0.25 s, n ? 2; hSOD1G93A? 1.03 ? 0.27 s,
n ? 5; p ? 0.026) currents were signifi-
cantly decreased (Fig. 5A, traces; Fig. 5C
for distribution of means), whereas mean
amplitudes of sAMPA (mSOD1WT?
12.3 ? 1.0 pA; hSOD1G93A? 17.4 ? 3.3
pA; p ? 0.21) and sIPSC (mSOD1WT?
14.1 ? 6.5 pA; hSOD1G93A? 25.1 ? 6.1
pA; p ? 0.35) currents were not signifi-
cantly different (Fig. 5A, traces; Fig. 5B for
distribution of means). Similar to SC in-
terneurons, changes in synaptic event fre-
quency were attributable to increased
activity-dependent release, because HM
mAMPAR mean amplitude (mSOD1WT
? 16.5 ? 1.9 pA, n ? 5; hSOD1G93A?
16.6 ? 2.4 pA, n ? 7; p ? 0.97) and inter-
event interval (mSOD1WT? 2.38 ?
0.46 s; hSOD1G93A? 1.96 ? 0.55 s; p ?
0.60) were unchanged (Fig. 5B,C) in P4–
P10 hSOD1G93AHMs compared with
mSOD1WT. Also, as in SC interneurons,
2.1 pA, n ? 3; hSOD1G93A? 15.6 ? 2.4
pA, n ? 5; p ? 0.92), interevent interval
(mSOD1WT? 7.6 ? 2.4 s; hSOD1G93A?
29.15 ? 14.0 s; p ? 0.29), and rise time
7.0 ? 0.7 s; p ? 0.41) of sNMDA currents were unchanged (Fig.
5B–D for distributions of means), whereas sNMDA events de-
cayed faster (Fig. 5E for distributions of means) (mSOD1WT?
80.4 ? 5.8 s; hSOD1G93A? 40.8 ? 7.7 s; p ? 0.012), suggesting
that an accelerated developmental switch from NMDAR NR2B
subunits to NR2A subunits was also present in motoneurons.
Although the normal development of synaptic inputs to HMs is
not well characterized, the similarity between changes in sponta-
neous synaptic activity in SC interneurons and HMs in
hSOD1G93Amice suggests that a similar acceleration of synaptic
development is occurring in the neuromotor system.
As enhanced excitatory and inhibitory synaptic activity and
changes in sNMDA decay kinetics in SC interneurons and HMs
( p?0.01)andsGABA( p?0.05)currentsaresignificantlyincreased(i.e.,intereventintervalisdecreased)inhSOD1G93Amice,
whereas amplitude, rise time, and decay time of these current types are not significantly different. There are no significant
vanZundertetal.•NeonatalHyperexcitabilityinMouseALSModelJ.Neurosci.,October22,2008 • 28(43):10864–10874 • 10869
suggest that the hSOD1G93Amutation triggers precocious matu-
ration during early postnatal development (Aamodt et al., 2000;
Shi et al., 2000; van Zundert et al., 2004), we sought to obtain
additional evidence in support of this hypothesis in the neuro-
whether structural maturation is faster in hSOD1G93Amice, we
retrogradely labeled HMs by tongue injection of herpes simplex
P0–P1 hSOD1G93Aand mSOD1WTlittermates (Fig. 6A–C)
(Neve et al., 2005; Olson et al., 2005). We found that structural
maturation of HM dendritic architecture is detectable as with-
drawal of HM dendrites crossing the brainstem midline between
P6 and P9 (Fig. 6D,E) (cf. Nu ´n ˜ez-Abades et al., 1994). Next we
analyzed P6 brainstem sections prepared from mSOD1WTand
hSOD1G93Amice from the same litter (Fig. 6F,G). As can be
observed, HMs from hSOD1G93Amice (Fig. 6G) showed signifi-
cantly fewer dendrites crossing the midline at P6 compared with
control littermates (Fig. 6F) (mean number of dendrites in
mSOD1WT? 1.88 ? 0.19, n ? 5; in hSOD1G93A? 0.17 ? 0.02,
n ? 3; unpaired t test, p ? 0.0006).
Our observations of faster structural maturation (Fig. 6), in-
creased intrinsic hyperexcitability (Figs. 1, 2), and altered synap-
tic transmission mediated by AMPA, NMDA, GABAA, and gly-
cine receptors (Figs. 4, 5) of SC interneurons and HMs indicate
perturbed at an early postnatal age. To investigate whether alter-
tor development, we studied six different behavioral responses
(Amendola et al., 2004) in hSOD1G93A, hSOD1WT, and
superimposedovertheindividualcellvalues.NotethatthefrequencyofsAMPA( p?0.01)andsIPSC( p?0.05)currentsaresignificantlyincreased(i.e.,intereventintervalisdecreased)inhSOD1G93Amice,
10870 • J.Neurosci.,October22,2008 • 28(43):10864–10874 vanZundertetal.•NeonatalHyperexcitabilityinMouseALSModel
development of forelimb placing (Fig. 7A) was transiently re-
duced in P3–P4 hSOD1G93Amice compared with both
mSOD1WTand hSOD1WTmice ( p ? 0.05, ?2test). In contrast,
neither forepaw nor hindpaw grasping (Fig. 7C,E) were signifi-
cantly different between genotypes. These results suggest that
development of the gross locomotor ability of the forelimb was
In addition, we also found that the righting response was tran-
siently reduced in P2 hSOD1G93Amice (Fig. 7B) ( p ? 0.05),
impaired. No differences were observed in cliff-drop aversion
and vibrissae placing (Fig. 7D,F), suggesting that maturation of
labyrinthine function and of vibrissae sensibility was normal in
hSOD1G93Amice compared with mSOD1WTand hSOD1WT
which upper and lower motoneurons are
electrophysiological, anatomical, and be-
havioral data from neonatal hSOD1G93A
mice provide conclusive evidence that
neural function is already abnormal dur-
ing early development in this mouse
model of FALS, occurring some 2–3
months before motoneurons degenerate
and clinical symptoms appear. Moreover,
we have shown that these early functional
changes in neural and synaptic activity are
present in interneurons. Together, our
findings suggest a hypothesis for the etiol-
ogy of FALS, in which the neonatal onset
of generalized CNS hyperactivity and ac-
celerated development may be compen-
adult clinical symptoms when aging or
ability and increased PCNain presumptive
bryonic hSOD1G93Amice (Kuo et al.,
and the consequent loss of trophic feed-
back to embryonic motoneurons from
Banks et al., 2005), or alterations in the
neuronal environment, such as interac-
tions with glial cells (Clement et al., 2003;
Boille ´e et al., 2006; Di Giorgio et al., 2007;
Nagai et al., 2007), are potentially a source
of significant alterations in motoneuron
activity and survival in these preparations.
Although acute brain slice preparation
causes axotomy, which can decrease syn-
sion in motoneurons (Moran and Grae-
ber, 2004), these responses do not develop
until at least 12–24 h after axotomy
recordings have been made.
The correlation between increased firing rate and increased
This is supported by our finding that riluzole, which selectively
AP amplitude in HMs and SC interneurons in hSOD1G93Amice
suggested that Na?channel density might significantly increase
imal rate of rise, a parameter linearly coupled to total Na?chan-
nel density (Kole et al., 2008), found no significant increase in
either HMs or SC interneurons, ruling out increased Na?chan-
nel density as a possible cause of increased PCNaand AP
As other developmental changes in motoneurons are acceler-
Low-magnification differential interference contrast (B) and fluorescent confocal (C) images of 350-?m-thick 4%
HMs dendrite retraction occurs earlier in presymptomatic hSOD1G93Amice (P6). A, Schematic of HSV-GFP p1003
vanZundertetal.•NeonatalHyperexcitabilityinMouseALSModel J.Neurosci.,October22,2008 • 28(43):10864–10874 • 10871
ated in hSOD1G93Amice, one possible ex-
planation for increased PCNawithout in-
creased total Na?channel density is that
the normal change-over in Na?channel
isoforms seen in developing motoneurons
occurs earlier in hSOD1G93Amice. Func-
tionally, embryonic motoneuron excit-
ability is initially driven by NaV1.2 and 1.3
expression, but subsequent increases in
within the first 3 weeks after birth (Porter
et al., 1996; Alessandri-Haber et al., 2002).
The fraction of PCNaproduced by Na?
channel isoforms differs, with NaV1.2 and
1.3 producing a small PCNa(?1% of total
Na?current), whereas NaV1.1 and 1.6
current) (Goldin, 1999). Thus, increased
PCNacould reflect the precocial appear-
ance of Na?channel isoforms with en-
hanced levels of persistent activity.
Other possible mechanisms for in-
creased PCNaalso merit consideration.
Oxidative stress can increase activity of
several protein kinases (Facchinetti et al.,
1998) capable of phosphorylating Na?
of Na?channel activity. Protein kinase C
(PKC) is of particular interest, because
PKC activity is induced both by activity-
mitochondrial generation of reactive oxi-
dation species (Facchinetti et al., 1998;
Knapp and Klann, 2000; Hu et al., 2003a;
Hongpaisan et al., 2004), because of direct
oxidation of thiol groups on the PKC pro-
tein (Knapp and Klann, 2000). PKC activ-
ity is elevated in spinal cord tissue from
hSOD1G93Amice and from human ALS patients (Hu et al.,
2003a,b). PKC-mediated phosphorylation of Na?channels in-
creases channel open time and decreases channel inactivation
oxidative stress and Ca2?entry driven by activity-dependent ac-
tivation of voltage-gated Ca2?channels could activate PKC, in-
creasing PCNaand motoneuron excitability. The resulting in-
crease in Ca2?
influx and oxidative stress because of
mitochondrial metabolic demands (Facchinetti et al., 1998)
could then further activate PKC in a vicious feedback cycle. Ex-
buffering capacity, so that excess Ca2?is stored in mitochondria
(von Lewinski and Keller, 2005), eventually leading to structural
and functional damage. Mitochondrial structure is already ab-
normal in motoneurons in 2- to 3-week-old hSODG93Amice
(Bendotti et al., 2001), and subsequent mitochondrial break-
down and activation of caspases (Li et al., 2000) may trigger mo-
are dependent on enhanced plasticity during a limited postnatal
“critical period” (Turrigiano and Nelson, 2004). Essential ele-
ments in this plastic period include activity-dependent remodel-
ing of receptor phenotype and neuronal morphology (van
Zundert et al., 2004; Turrigiano and Nelson, 2004). Our data
indicate that these elements are perturbed in neonatal
hSOD1G93Amice, consistent with our observation of enhanced
neuronal activity. In both motoneurons and interneurons, we
observed faster decay kinetics in NMDA glutamate receptor-
composition from NR2B-containing to NR2A containing recep-
tors. This subunit switch has been well characterized in SC inter-
neurons, and is normally closely dependent on retinal activity-
driven synaptic inputs as the retina matures and on eye opening
(Shi et al., 2000; Townsend et al., 2003; Lu and Constantine-
Paton, 2004). Our data are consistent with the triggering of an
interneurons, presumably because of enhanced synaptic activity
even in the absence of increasing retinal inputs. Although the
normal development of NMDA receptor responses is less well
position is also apparent. We also shown that the extensive re-
hSOD1G93Amice. In motoneurons and other neurons, dendritic
remodeling during development is regulated by consequences of
neural activity, including NMDA and AMPA receptor activation
(Kalb, 1994; Inglis et al., 2002), Ca2?influx, and nitric oxide
release (Xiong et al., 2007). All of these factors will be enhanced
P2–P4. The fraction of pups displaying forelimb placing (A, at P3–P4 only) or righting (B, at P2 only) responses was lower in
Transient delays in development of gross locomotor abilities in presymptomatic hSOD1G93Amice are present at
10872 • J.Neurosci.,October22,2008 • 28(43):10864–10874vanZundertetal.•NeonatalHyperexcitabilityinMouseALSModel
by hyperactivity in hSOD1G93Amice, thus accelerating the nor-
mal time course of dendrite remodeling.
Together, we show here that during very early development
(P4–P12) the hSOD1G93Amutation is associated with hyperex-
citability (Figs. 1, 3) and increased activity-dependent excitatory
neurons. In motoneurons, hyperexcitability is associated with
abnormally large PCNa(Fig. 2). These changes are the earliest
potentially pathogenic events yet described in the hSOD1G93A
onset of limb and tongue motor weakness at 2–3 months, and
al., 2001; Smittkamp et al., 2008). These results are consistent
with the hypothesis that increased persistent Na?channel activ-
ity, causing widespread hyperexcitability and enhanced synaptic
activity in the developing CNS, is a possible mechanism leading
and Kiernan, 2006; Vucic et al., 2008). However, further work is
required to determine whether widespread increases in neuronal
and synaptic activity are also present in other genetic mutations
associated with FALS (Pasinelli and Brown, 2006) or in sporadic
tral neurons of hSOD1G93Amice are more active suggests that
this may reflect the attainment of a homeostatic balance in op-
posing synaptic activities (Turrigiano and Nelson, 2004) to ac-
tively compensate for generalized neuronal hyperexcitability.
The structural changes in HM morphology (Fig. 6) and the tran-
sient disruption of some developing motor behaviors (Fig. 7)
before the establishment of a homeostatic state in the neuromo-
tor system. Early network changes, without the loss of neurons,
may also occur in other genetically induced adult neurodegen-
erative diseases, such as Alzheimer and Parkinson’s disease
This evidence suggests a novel hypothesis for the adult onset of
overt FALS symptoms, namely that they result from age-related
factors (e.g., neuron loss or other traumatic insults) that cause a
early onset of a generalized hyperexcitability and increased syn-
aptic activity in the neonatal hSOD1G93Amouse, a mechanistic
link between these phenomena and neuronal death in the adult
will determine whether the onset of motoneuron death in this
animal model of FALS might be prevented or mitigated by sup-
pression of neuronal hyperactivity in early life.
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