Cerebral Cortex March 2011;21:574--587
Advance Access publication July 12, 2010
Blocking Early GABA Depolarization with Bumetanide Results in Permanent Alterations
in Cortical Circuits and Sensorimotor Gating Deficits
Doris D. Wang1,2and Arnold R. Kriegstein1,3
1Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco CA 94143,
USA2Medical Scientist Training Program, University of California, San Francisco, CA 94143, USA and3Department of Neurology,
University of California, San Francisco, CA 94143, USA
Address correspondence to Dr Doris D. Wang, 513 Parnassus Avenue, UCSF Box 0525, San Francisco, CA 94143, USA. Email: firstname.lastname@example.org.
A high incidence of seizures occurs during the neonatal period when
immature networks are hyperexcitable and susceptible to hyper-
syncrhonous activity. During development, g-aminobutyric acid
(GABA), the primary inhibitory neurotransmitter in adults, typically
excites neurons due to high expression of the Na1-K1-2Cl2
cotransporter (NKCC1). NKCC1 facilitates seizures because it
renders GABA activity excitatory through intracellular Cl2accumu-
lation, while blocking NKCC1 with bumetanide suppresses seizures.
Bumetanide is currently being tested in clinical trials for treatment
of neonatal seizures. By blocking NKCC1 with bumetanide during
cortical development, we found a critical period for the development
of a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate synapses.
Disruption of GABA signaling during this window resulted in
permanent decreases in excitatory synaptic transmission and
sensorimotor gating deficits, a common feature in schizophrenia.
Our study identifies an essential role for GABA-mediated de-
polarization in regulating the balance between cortical excitation
and inhibition during a critical period and suggests a cautionary
approach for using bumetanide in treating neonatal seizures.
Neonatal seizures are among the most common pathological
manifestations seen in the developing brain, affecting 1.5 to
5.5 per 1000 newborns each year (Lanska et al. 1995; Ronen
et al. 1999; Saliba et al. 1999). Seizures occur more often in the
neonatal period than any other time in life because immature
networks have a propensity for generating synchronized
activity. Unlike seizures in adults, seizures in neonates respond
poorly to anticonvulsants that work by enhancing the opening
of c-aminobutyric acid (GABA)Achannels (Painter et al. 1999;
Kahle and Staley 2008). GABA is the principal inhibitory
neurotransmitter in the adult brain and exerts a hyperpolarizing
effect on the membrane potential by opening the GABAA
chloride (Cl–) channel and also GABABreceptors (Owens and
Kriegstein 2002; Ben-Ari 2006; Wang and Kriegstein 2009).
However, in the developing brain, GABA excites immature
neurons due to an elevated intracellular Cl–concentration and
a depolarizing Cl–equilibrium potential, which may account for
neonates’ resistance to anticonvulsant drug therapy (Ben-Ari
and Holmes 2005; Kahle and Staley 2008).
The excitatory effects of GABA depend upon the expression
of the Na+-K+-2Cl–cotransporter (NKCC1), which accumulates
Cl–in cortical neurons from the embryonic stage until early
postnatal life in both rodents and humans (Plotkin et al. 1997;
Wang et al. 2002; Dzhala et al. 2005). On the other hand, the
K+-Cl–cotransporter (KCC2) exports Cl–out of the cell and is
weakly expressed at birth and upregulated as the nervous
system matures (Rivera et al. 1999; Li et al. 2002). NKCC1 and
KCC2 have been linked to impaired Cl–regulation and epilepsy.
NKCC1 facilitates drug-induced seizures in the developing
brain (Dzhala et al. 2005), and KCC2-hypomorphic mice
exhibit hyperexcitable hippocampal CA1 neurons and are
more prone to epilepsy (Woo et al. 2002; Tornberg et al. 2005).
In addition, loss of KCC3, another isoform of the K–-Cl–
cotransporter mutated in human Anderman syndrome, is
associated with reduced seizure threshold in mice (Boettger
et al. 2003). Interestingly, quantitative reverse transcriptase
polymerase chain reaction analysis of patients suffering from
drug-resistant temporal lobe epilepsy reveal an upregulation of
NKCC1 messenger RNA (mRNA) and a downregulation of
KCC2 mRNA in the hippocampus (Palma et al. 2006).
Recent studies demonstrate that by using the selective
antagonist bumetanide to inhibit NKCC1, certain forms of
epileptiform activity can be suppressed in vitro and in vivo
(Dzhala et al. 2005; Kilb et al. 2007). While these studies show
great potential for using bumetanide to treat neonatal seizures,
there are significant questions that must first be addressed;
namely, does inhibiting NKCC1 have deleterious effects in the
developing nervous system? Cortical synapse formation begins
from the late embryonic stage (E18 in rats) until the first few
weeks of postnatal life in rodents, during which newborn
neurons express GABAA receptors and receive GABAergic
inputs before forming glutamatergic synapses (Owens et al.
1999; Tyzio et al. 1999; Hennou et al. 2002; Ben-Ari 2006).
Recently, studies have shown that GABA-mediated depolariza-
tion can regulate the development of excitatory synapses in the
developing cortex in vivo (Wang and Kriegstein 2008).
However, the long-term effects of inhibiting NKCC1 on cortical
synapse formation remain unclear. Here, we show that
bumetanide treatment during a critical developmental period
in mice results in lasting disruption of cortical excitatory
synapse formation and that this disturbance in the ratio of
excitatory and inhibitory inputs in cortical neurons leads to
behavioral abnormalities in adult mice.
Materials and Methods
Timed-pregnant Swiss-Webster dams were obtained from Simonsen
Laboratories, Gilroy, CA. All manipulations were performed in
accordance with the guidelines of the UCSF Institutional Animal Care
and Use Committee.
Intraperitoneal injections were given to pregnant dams prenatally and
their pups postnatally. Injections were placed into the lower abdomen
with slight retraction of the injection needle to ensure that the drugs
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are not directly delivered to the embryos. The following drugs were
dissolved in phosphate-buffered saline (PBS) and injected at the
following doses: bumetanide (Sigma) 0.2 mg/kg and hydrochlorothia-
zide (HCTZ, Sigma) 4 mg/kg. Drugs were diluted to concentrations so
that the volume of injection (mL) = weight in kg 3 10.
Drug-treated postnatal mice at different ages were anesthetized and
processed for slice preparation as previously described (Owens et al.
1996). Neonatal brains were quickly removed into ice-cold artificial
cerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 5 KCl,
1.25 NaH2PO4, 1 MgSO4, 2 CaCl2, 26 NaHCO3, and 10 glucose
oxygenated with 95% O2--5% CO2 (pH 7.4). Neonatal brains were
embedded in 4% low melting point agarose in ACSF, hardened on ice,
and cut into coronal slices (300 lm thick) using a vibratome
(Leica VT1000s). Older animals were anesthetized with avertin
(0.5--0.75 mg/g) and perfused with ice-cold solution containing (in
mM) 248 Sucrose, 5 KCl, 1.25 NaH2PO4, 1 MgSO4, 2 CaCl2, and
26 NaHCO3prior to brain removal in ACSF. Brains from older animals
were cut directly on the vibratome, and sections are confined to the
dorsal regions of the cortex. Electrophysiological recordings were
obtained at room temperature (RT) from sections continuously
perfused with oxygenated ACSF. For recordings at 32 ?C, an inline
heater was used to control the temperature of the bath solution
(Warner SC-20). Pyramidal neurons were visualized by microscopy
under differential interference contrast and identified by their
morphology, location within layer II/III of cortex, and current profile
in response to 20-mV voltage steps (Noctor et al. 2004; Wang and
Kriegstein 2008). Microelectrodes (6--8 MX resistance) were pulled
from borosilicate glass capillaries (Cornig 7056 Thin Wall) using
a 2-step pipette puller (Narishige) and filled with the following solution
(in mM): 130 KCl, 5 NaCl, 0.4 CaCl2, 1 MgCl2, ten 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (pH 7.3), 11 ethylene glycol tetraacetic
acid, and 0.1% Lucifer yellow for majority of the recordings. For
perforated patch recordings, the gramicidin stock (10 mg/mL in
dimethyl sulfoxide) was diluted in the internal pipette solution above
to a final concentration of 25 lg/mL. Tip of the pipette was filled with
the gramicidin-containing solution and the rest with normal internal
pipette solution. Gigaohm seals were made, and cells were applied
voltage ramps from --140 to +100 mV every 20 s to monitor current
changes. After ramp currents stabilized, 3 s focal application of 100 lM
GABA (Sigma) was applied using the DAD superperfusion (pressure
drug application) system (ALA Scientific) to activate GABAARs on
neurons. Voltage ramps from --140 to +100 mV were applied before and
after GABA application. The resulting currents were then subtracted,
and the difference plotted as I--V curves (Fig. 1A,B). EGABA was
determined for each cell as x-intercept of I--V curves. To characterize
miniature postsynaptic currents (mPSCs), 5--20 min of whole-cell
recordings were made using a Double Patch-clamp EPC9/2 (HEKA)
with 10 kHz data acquisition frequency. The series resistances were
monitored and only those that changed less than 20% during experi-
ments were used for data analysis. Traces were analyzed using
MiniAnalysis (Synaptosoft) program. Each synaptic event was manually
selected based on rise time, amplitude, and decay properties. Additional
drugs were added as indicated with the following estimated final
concentrations: bicuculline methiodide (100 lM, Sigma), DNQX
(20 lM, Tocris), tetrodotoxin (TTX, 0.5 lM, CalBiochem). To record
N-methyl-D-aspartic acid (NMDA) channel currents, recordings were
performed in ACSF containing TTX (0.5 lM), bicuculline methiodide
(100 lM), 6,7-Dinitroquinoxaline-2,3-dione (DNQX) (20 lM), and
Glycine (40 lM, Sigma). Unless otherwise noted, the holding potential
for voltage clamp recordings was –70 mV.
To calculate the frequency of excitatory postsynaptic current (EPSC)
and inhibitory postsynaptic currents (IPSCs), cells were recorded for
1--2 min to establish the baseline mPSC frequency. Focal application of
bicuculline (100 lM) was applied for 1 min to isolate the EPSC
frequency, and IPSC frequency is calculated by subtracting the EPSC
frequency from the baseline mPSC frequency. Whenever possible,
DNQX (20 lM) was applied after washout of bicuculline to isolate the
IPSC frequency. Application of both bicuculline and DNQX abolished
all mPSCs in our recording conditions. To calculate the inhibition--
excitation ratio, we used the ratio of IPSC to EPSC frequencies for each
In Utero Injection and Electroporation of Plasmids
Plasmids expressing green fluorescent protein (GFP) or yellow
fluorescent protein (YFP) under the phosphoglycerate kinase (PGK)
promoter were introduced into the in vivo developing cortex by
intraventricular injection and electroporation (Saito and Nakatsuji
2001). The PGK gene encodes for the constitutively active glycolytic
enzyme PGK and its promoter is used in high-expression vectors
(Graham and Chambers 1997). Intraventricular injections were carried
out in E15 in timed-pregnant Swiss-Webster mice as previously
described (Noctor et al. 2001). Electroporations were performed using
an Electro Square Porator ECM830 (Genetronics) (5 pulses, 45 mV,
100 ms, 1-s interval). One microliter of DNA was injected per brain
at a concentration of 1.5 lg/lL.
Electroporated postnatal mice were perfused as previously described,
and 300-lm-thick coronal sections were cut using a vibratome (Leica
VT1000S) and stored in PBS. Images of GFP+or YFP+pyramidal neurons
were acquired on a Leica TSC SP5 laser-scanning confocal microscope.
For morphology analysis of the GFP+pyramidal neurons, 3D recon-
structions of the cell body and dendritic processes were made from
Z-series stacks of confocal images using the Imaris software. All
dendrite properties were quantified using the filament function from
Imaris, which constructs a 3D representation of each segment of the
dendrite with a series of cylinders or truncated cones. Dendrite volume
is defined as the sum of the volumes of all dendrite segments. For soma
area analysis, the images were collapsed and the cell body was
semiautomatically traced with NIH ImageJ. Spine densities were
calculated by counting the number of spines on a high magnification
collapsed Z stack and dividing the total spine number by the length of
dendrite. Only bright protrusions adjacent to or contiguous with the
dendrites are counted as spines. Dim blebs were not counted. Spines
were discriminated from one another when discrete edges are
detected between the individual spines.
The protocols were adapted from previous study by Scearce-Levie et al.
(2008). Dams were treated with PBS or bumetanide beginning on
gestational day 15 and their pups treated until postnatal day 7.
Beginning on P2, pups were individually numbered to blind the
experimenter and tested daily for developmental milestones appropri-
ate for the age. Early milestone testing ranged from P2 to P9 and
consisted of surface righting, grasp reflex, cliff avoidance, and negative
geotaxis. Later milestone testing ranged from P10 to P17 and consisted
of visual placing, bar hanging, and air righting. Pups were picked at
random and tests were performed in the order stated above, from the
least to most traumatic (i.e., performing tests that involve falling at the
end of each session) to avoid carryover effects. Unless otherwise noted,
absence of a milestone was scored if the mouse did not exhibit the
behavior within 60 s on the first try. During testing, all pups were
transferred to a cage filled with clean bedding. Individual pups were
removed, weighted, and checked for physical abnormalities including
head and body deformities. To assess surface righting, each pup was
placed on its back and monitored until it successfully righted itself. To
assess negative geotaxis, the pup was placed facing downward on
a sheet of textured plastic inclined at a 30? angle and monitored until
the pup reoriented itself with its head and forelimbs higher up the
plane than its hindlimbs. To assess cliff avoidance, the pup was placed
with its hindlimbs resting on a circular Styrofoam platform mounted on
a 30-cm high stand. The pup was positioned so its forepaws and nose
were suspended over the edge of the platform and monitored until the
pup moved away from the edge. To assess grasp reflex, each forepaw
was gently stroked with the wooden end of a swab. If the pup
Cerebral Cortex March 2011, V 21 N 3 575
immediately curved its paw to grasp the swab, the grasp reflex was
considered present. The date when both eyes were open was recorded.
Visual placing was assessed by suspending the pup by its tail and gently
lowering it toward the tabletop. If the pup raised its head and extended
forelimbs toward the surface, visual placing was scored as present. To
assess air righting, the pup was held with its ventral side facing upward
30 cm above a chamber filled with soft bedding. The pup was released,
and air righting was considered present if the pup turned while falling
so that it landed on its feet. To assess bar hanging, the pup was allowed
to grasp a small wire bar and then released so that it was hanging by its
forelimbs. Once the pup was able to hang suspended for 10 s, bar
holding was scored as present.
Figure 1. Bumetanide hyperpolarizes GABA reversal potential in immature neurons (A) and (B) voltage-ramps obtained from P0 cortical neurons of pups treated with
intraperitoneal injections of either saline or bumetanide under gramicidin-perforated patch-clamp conditions. EGABAreversed at more hyperpolarized potentials in bumetanide-
treated mice than in controls (as indicated by the x-intercept). (C) Blocking NKCC1 with bumetanide effectively changed the chloride gradient in newborn cortical neurons, making
GABA hyperpolarizing to the resting potential instead of depolarizing as in control cells. Bar graphs indicate mean ± SEM (number of recorded cells are indicated in parenthesis in
bar graphs, n 5 3 animals per condition,***P\0.0001, t-test). (D) Comparison of EGABAvalues for control- and bumetanide-treated cells recorded at RT and at 32 ?C. Increasing
the recording temperature did not significantly alter the EGABA. Bar graphs indicate mean ± SEM (number of recorded cells are indicated in parenthesis in bar graphs, n 5 2
animals at 32 ?C, ***P\0.0001, t-test). (E) Timeline of different drugs treatments. Black line represents length of control (PBS) treatment, and colored lines represent different
bumetanide (Bum) exposure windows.
Bumetanide Alters Cortical Circuits and Sensorimotor Gating Functions
Wang and Kriegstein
The protocols were adapted from a previously study, and experi-
menter was blinded to the condition (Scearce-Levie et al. 2008).
Mice were tested in a 3-chambered box with small openings in the
dividing walls allowing access from the center chamber into left and
right chambers. Each chamber was cleaned and fresh bedding added
between trials. The paradigm consisted of a 3-stage procedure.
Stage1: habituation; test mouse was first placed in the center
chamber and allowed to explore all 3 chambers of the apparatus for
5 min. Stage 2: Sociability; an unfamiliar mouse (male C57/Bl6
mouse) was placed in either the left or right chamber enclosed in
a small internal wire cage; placement of Stranger 1 in the left or right
chamber alternated between trials, with an empty but otherwise
identical wire cage in the opposite chamber. Following placement of
Stranger 1 into the left or right chamber, the test mouse was allowed
to leave the center chamber and explore all 3 chambers of the
apparatus for 10 min, and time spent in each compartment was
recorded. Stage 3: Novelty; with the initial stranger (now familiar)
retained in its original chamber, a second, unfamiliar mouse (novel,
male C57/Bl6 mouse) was placed in the previously empty wire cage
in the opposite chamber. The test mouse was allowed to leave the
center chamber and explore all 3 chambers of the apparatus for
a second period of 10 min, and time spent in each compartment was
Testing was conducted as previously described, and experimenter was
blinded to the condition (Cheng et al. 2007). Activity in the open field
was tested with the automated Flex-Field/Open Field Photobeam
Activity System (PAS; San Diego Instruments). The system consisted of
2 identical clear plastic chambers (41 3 41 3 38 cm), a PAS control box,
a PC interface board, and a microcomputer for recording and analysis of
data. Two sensor frames, each consisting of a 16 3 16 photobeam array
at 1.5 and 6 cm above the bottom of the cage, were used to detect
movements in the horizontal and vertical planes. The test was initiated
by placing the mouse in the center of the arena. Horizontal beam
breaks (ambulatory moves) in the arena were counted more than
15 min. The arena was cleaned and dried after each test. The
illumination level of the box was 271 lux.
Elevated Plus Maze
Testing was conducted as previously described, and experimenter was
blinded to the condition (Cheng et al. 2007). The elevated plus-shaped
maze consisted of 2 open arms and 2 closed arms equipped with rows
of infrared photobeams (Hamilton-Kinder). Mice were habituated to
dim lighting in the testing room for 30 min and then were placed
individually at the center of the apparatus and allowed to explore for
10 min. The time spent and distance traveled in each of the arms were
recorded by infrared beam breaks. After each mouse was tested, the
apparatus was thoroughly cleaned.
Startle Reactivity and Prepulse Inhibition
Acoustic startle reactivity and prepulse inhibition (PPI) were measured
as described, and experimenter was blinded to the condition (Esposito
et al. 2006). Acoustic startle reactivity was measured with 2 identical
startle chambers (Hamilton-Kinder) containing a transparent non-
restrictive plexiglas box resting on a platform inside a soundproof
ventilated box. An acoustic speaker generating broadband bursts was
mounted 15 cm above the box produced all the acoustic stimuli. Mouse
movements were detected and transduced by a piezoelectric acceler-
ometer mounted under each cylinder. Movements were digitized and
stored by a computer and interface assembly. Movements were moni-
tored for 65 ms after the onset of each stimulus, and the maximum
amplitude response (Newtons) was used to determine the startle
response. All tests were performed during the same phase of the light
cycle, between the 12-h light cycles from 8 AM to 8 PM.
For testing, mice were placed inside the soundproof chamber. After
a 5-min acclimation period, each session consisted of 80 trials of
5 types: 24 trials of a 40 ms, 120 dB (sound pressure level scale) startle
stimulus alone (to measure maximum acoustic startle amplitude);
14 trials without startle stimulus (to measure baseline movements in
the chamber); and 14 trials each of a 40-ms stimulus at 4, 8, or 16 dB
above the 65 dB background (prepulse), followed by a 100-ms interval
and a 40 ms 120-dB startle stimulus. In the first 5 and the last 5 trials of
each session, the startle stimulus alone was presented to determine the
degree of habituation to the startle stimulus. The other trials of startle
stimulus alone and prepulse plus startle stimulus were presented
in pseudorandom order with an average intertrial interval of 15 s
(range: 7--23 s).
The percentage of PPI of the startle response was calculated as
follows: 100 -- ([average response to prepulse plus startle stimulus/
average response to startle stimulus alone] 3 100). Thus, a high value
indicates high PPI reflected by a large reduction in startle response
when the prepulse preceded the startle stimulus.
Group measures are expressed as mean ± standard error of the mean
(SEM); error bars also indicate SEM. Prism software (version 4.0) was
used for statistical analysis. We assessed the statistical significance or
differences between control and experimental conditions with
a 2-tailed Student’s t-test, a one-way analysis of variance (ANOVA) for
3 or more groups and a 2-way repeated measure ANOVA with posttest
for the PPI test. For developmental milestone tests, we used logrank
test to assess for statistical significance. Experimenters were blinded
during testing. Statistical significance was set at P < 0.05.
Bumetanide Negatively Shifts GABA Reversal Potential in
To test the effect of bumetanide on the GABA reversal potential
(EGABA), we gave daily intraperitoneal injections of either
bumetanide (0.2 mg/kg) (Dzhala et al. 2005) or PBS to pregnant
mice beginning at gestational day 15 (E15) until birth (P0). We
performed gramicidin-perforated patch recordings of P0
cortical neurons in acute brain slices from either bumetanide-
or saline-treated pups to confirm that bumetanide can shift
EGABA toward a hyperpolarizing potential. Perforated patch
recording allowed us to measure the intracellular Cl–concen-
tration without perturbing the Cl–gradient. At room temper-
ature, EGABA was significantly more negative in neurons of
bumetanide-injected animals than in controls (t-test, control vs.
bumetanide: –40.9 ± 2.8 mV vs. –65.8 ± 3.4 mV; P < 0.0001;
Fig. 1A--C). With the unaltered resting membrane potential
of cortical neurons at P0 (PBS: –50.9 ± 5.1 mV, bumetanide:
–51.1 ± 4.4 mV, P > 0.05), GABA hyperpolarized the neonatal
cortical neurons of bumetanide-treated mice, thus validating
the effectiveness of bumetanide in abolishing GABA-induced
depolarization (Fig. 1C). Because temperature can affect trans-
porter kinetics, we repeated our recordings at more physio-
logical temperatures. The EGABA values from recordings
performed at 32 ?C were not significantly different from those
recorded at room temperature of 25 ?C, which confirms the
efficacy of bumetanide to block NKCC1 in the brain (t-test,
control RT vs. 32 ?C: –40.9 ± 2.8 mV vs. –43.7 ± 0.7 mV; P >0.05;
bumetanide RT vs. 32 ?C: –65.8 ± 3.4 mV vs. –68.1 ± 0.9 mV;
P >0.05; Fig. 1D).
Blocking NKCC1 with Bumetanide during a Critical
Period Leads to Lasting Changes in Cortical Excitatory
A recurrent theme in neocortical development is the idea that
changes to sensory afferents during a critical period in
Cerebral Cortex March 2011, V 21 N 3 577
development will result in lasting alterations in cortical
organization. The formation of ocular dominance columns in
the visual cortex and barrels in the somatosensory cortex are
well-known examples of such critical periods (Hensch 2005;
Hensch and Fagiolini 2005). Because GABAergic signaling
provides the main excitatory drive during corticogenesis (until
the first postnatal week in rodents), the period of GABA
depolarization may provide a window for initial circuit
formation in the neocortex (Ben-Ari 2006).
To test the effect of bumetanide in cortical circuit formation,
we exposed pregnant mice and their pups to bumetanide at
different developmental windows (Fig. 1E). We used PBS
treatment from E15 to P7 and bumetanide treatment from P7
to 14 (after GABA becomes hyperpolarizing, (Rivera et al. 1999)
as controls. To measure synaptic connectivity of cortical
neurons, we recorded mPSCs in layer II/III pyramidal neurons
at a holding potential of --70 mV in the presence of TTX
(0.5 lM) to block action potentials. Even though pyramidal
neurons in layer II/III may not homogenous, we studied this
population because they are the main source of intracortical
synaptic connections, and our previous study showed that
depolarizing GABA is important for circuit formation of
these upper layer cortical neurons (Wang and Kriegstein
2008). To isolate the a-amino-3-hydroxyl-5-methyl-4-isoxazole-
propionate (AMPA) and GABAergic mPSCs, we bath applied the
GABAAreceptor antagonist bicuculline methiodide (100 lM) or
the non-NMDA glutamate receptor antagonist DNQX (20 lM),
respectively (Fig. 2A,B). In 4-week-old control animals, the
pyramidal neurons exhibited robust AMPA and GABA synaptic
innervation with approximately equal numbers of excitatory
and inhibitory mPSCs (PBS: AMPA mPSCs: 2.0 ± 0.2 Hz, GABA
mPSCs: 2.3 ± 0.3 Hz, n = 19, 3 animals; Fig. 2C). By contrast,
bumetanide treatment during the entire period when GABA is
normally depolarizing (E15--P7) resulted in a drastic 10-fold
reduction in the frequency of AMPA mPSCs (PBS vs. bumeta-
nide: 2.0 ± 0.2 Hz vs. 0.22 ± 0.07 Hz, bumetanide n = 16,
3 animals, P < 0.0001), while GABA mPSC frequency was
unchanged (PBS vs. bumetanide 2.3 ± 0.3 Hz vs. 2.4 ± 0.4 Hz,
bumetanide n = 16, 3 animals, P >0.05; Fig. 2C). Calculating the
ratio of inhibitory to excitatory synapses at 4 weeks revealed
that bumetanide-treated mice had a significantly larger ratio of
inhibitory mPSCs compared with the controls (PBS vs.
bumetanide E15--P7, 1.2 ± 0.2 vs. 10.6 ± 3.9, P = 0.0113;
Fig. 2C). This defect in forming excitatory synaptic inputs
persisted to adulthood and resulted in a large ratio of GABA to
AMPA mPSCs (PBS vs. bumetanide E15--P7, 0.98 ± 0.22 vs. 2.68 ±
0.17, P < 0.0001; Fig. 2D).
The outcome of bumetanide treatment on AMPA and GABA
synaptic transmission remained the same in recordings
performed at 32 ?C, suggesting that the effects occur at
physiological temperatures (Supplementary Fig. 1A,B). Further-
more, bumetanide treatment had no effect on the amplitude
and kinetics of the AMPA and GABA mPSCs, suggesting that the
decrease in AMPA frequency likely reflects the decrease in
synaptic input rather than changes in AMPA receptor signaling
(Supplementary Fig. 1C--F). The decrease in AMPAergic trans-
mission was not accompanied by defects in NMDA receptor
signaling, as NMDA mPSCs isolated by recording in 0 Mg2+bath
solution containing bicuculline and DNQX revealed unaltered
frequency and amplitudes (Supplementary Fig. 2A--E).
To determine the minimal window during which bumeta-
nide treatment would result in permanent changes in cortical
excitatory synaptic transmission, we exposed mice to bume-
tanide at different developmental windows (E15--E19, E15--P5,
E17--P7, P0--P7, and P7--P14; Fig. 1E). Neither prenatal
treatment (E15--E19) nor postnatal treatment (P0--P7 and
P7--P14) with bumetanide resulted in significant changes in
AMPA or GABA mPSC frequencies (Fig. 2C,D). However,
bumetanide injections from E17 to P7 produced similar effects
as treatment from E15 to P7 (Fig. 2C,D). Interestingly, it
appears that bumetanide treatment from E17 to P7 resulted in
a transient decrease in GABA mPSC frequency at 4 weeks (PBS
vs. E17--P7: 2.3 ± 0.3 Hz vs. 0.9 ± 0.4 Hz, E17--P7: n =7, 2 animals;
P = 0.0480). However, it is unclear whether this decrease in
GABA mPSC was meaningful as this difference barely reached
statistical significance and disappeared by adulthood. When
examining the ratio of GABAergic to AMPA mPSCs, the control
conditions had a near 1:1 ratio of inhibitory to excitatory
synapses, but bumetanide exposure from E15 to P7 and E17 to
P7 significantly increased the ratio both at 4 weeks and in
adults (4 weeks: PBS vs. E17--P7: 1.16 ± 0.19 vs. 2.46 ± 0.66,
P = 0.0247; adult: PBS vs. E17--P7: 0.98 ± 0.23 vs. 1.78 ± 0.17,
P = 0.0064; Fig. 2C,D). Together these data suggest that
systemic blockade of NKCC1 with bumetanide during the
period of GABA depolarization permanently disrupts excitatory
synapse formation in the cortex, resulting in an abnormal
balance between excitatory and inhibitory inputs in the adult
Bumetanide Disrupts Morphology of Cortical Neurons
Disruptions in synaptic connections are often reflected
by changes in neuron morphology. Previous studies have
shown that AMPA receptor trafficking into developing
synapses is required to stabilize new dendritic branches
(Rajan and Cline 1998; Wu and Cline 1998; Shi et al. 1999;
Haas et al. 2006). To examine the effect of bumetanide
treatment on the morphology of cortical neurons, we
performed in utero injection and electroporation of plasmids
expressing either GFP or YFP into E15 mice embryos during
the period of generation of upper layer cortical neurons. We
injected the animals with either PBS or bumetanide from E15
to P7 and fixed and sectioned the brain at 4 weeks to take
confocal images of labeled neurons. At 4 weeks postnatal,
GFP+neurons from PBS- and bumetanide-injected animals had
typical pyramidal neuron morphology and indistinguishable
soma sizes (Fig. 3A--C). However, neurons from bumetanide-
treated animals exhibited fewer primary and secondary
dendrites compared with control cells (t-test, primary: PBS
vs. bumetanide, 6.9 ± 0.2 vs. 5.3 ± 0.2, P < 0.0001; secondary:
PBS vs. bumetanide, 11.9 ± 0.5 vs. 8.1 ± 0.3, P < 0.0001; PBS:
n = 16, 3 animals, bumetanide n = 48, 4 animals; Fig. 3D,E). We
semiautomatically traced the neurons to further evaluate the
effect of bumetanide treatment on morphology. We found
that compared with controls, bumetanide-treated cells had
significantly decreased dendrite length, dendrite volume,
branch levels, branch points, number of dendrite segments,
and terminal points (PBS: n = 17, 3 animals, bumetanide n =
18, 4 animals; t-tests, PBS vs. bumetanide, P < 0.0001 for all
parameters; Fig. 3F, Supplementary Fig. 3C--F). This extensive
impairment of dendrite formation in bumetanide-treated cells
is consistent with recent evidence showing that membrane
depolarization by GABA is critical for the morphological
maturation of cortical neurons (Cancedda et al. 2007; Wang
and Kriegstein 2008).
Bumetanide Alters Cortical Circuits and Sensorimotor Gating Functions
Wang and Kriegstein
To further elucidate the effect of systemic bumetanide
treatment on synapse formation, we measured dendritic
spines, which are postsynaptic sites of excitatory synapses.
We used confocal microscopy to analyze the mean spine
density on secondary dendrites of GFP+or YFP+cells from
animals treated with PBS or bumetanide from E15 to P7. We
found that the mean spine density in PBS-treated animals was
0.41 ± 0.01 spines/lm, whereas in the bumetanide-treated
Figure 2. Blocking NKCC1 with bumetanide during a critical period disrupts the balance of excitatory and inhibitory synapses in the adult. (A) and (B) current traces of mPSCs of
layer II cortical neurons recorded from 4-week-old and adult mice treated with either saline (PBS) or bumetanide from E15 to P7. Traces on the right represent expanded
segments of traces on the left. Cells were recorded at --70 mV holding potential and in ACSF containing 0.5 lM TTX. AMPA mPSCs were isolated with bath application of 100 lM
bicuculline and GABA mPSCs with 20 lM DNQX. (C) Frequency of AMPA (left) and GABA (middle) mPSCs for different windows of bumetanide exposure as described in
Figure 1D. Ratio of GABA to AMPA mSPCs (right) shows that bumetanide treatment from E15 to P7 and E17 to P7 resulted in significant increases due to defects in forming
excitatory AMPA synapses (D) same parameters analyzed in (C) but in 8- to 12-week-old adult mice. Bar graphs indicate mean ± SEM, number of recorded cells is indicated in
7, E15--E19 5 8, E15--P5 5 6, E15--P7 5 11, E17--P7 5 11, P0--P7 5 10, P7--P14 5 11; (*P\0.01, **P\0.001, ***P\0.0001 compared with control; t-test).
Cerebral Cortex March 2011, V 21 N 3 579
animals, the density was significantly decreased to 0.34 ± 0.01
spines/lm (PBS: n = 56, 3 animals; bumetanide n = 47, 4
animals; t-test, P < 0.001; Fig. 4A--C). The decreased spine
density in bumetanide-injected animals reflected the overall
decrease in presynaptic release events, as evidenced by the
decreased in AMPA mPSCs, confirming that blocking GABA-
mediated depolarization during a critical period disrupts
Figure 3. Bumetanidetreatmentaltersmorphologyofcorticalneurons.(A)and(B)3Dreconstructionsof2corticalneuronsfrom4-week-oldcontrolorbumetanide-treated(E15--P7)
mice. Animals were electroporated with GFP plasmids at E15 for visualization of the cells. Photographs on the left show flattened confocal stacks of neurons that
number of primary dendrites (D), numberof secondary dendrites (E), and total branching points (F) of traced cortical neurons. Compared withcontrol, bumetanide-treated pyramidal
neurons have less developed dendritic morphology. Scale bar, 20 lm. Bar graphs indicate mean ± SEM (PBS: n 5 3 animals, bumetanide n 5 4 animals; ***P\0.0001, t-test).
Bumetanide Alters Cortical Circuits and Sensorimotor Gating Functions
Wang and Kriegstein
Bumetanide Treatment Results in Developmental Delay
Abnormal cortical circuits have been implicated in many
neurological and psychiatric disorders including epilepsy,
obsessive-compulsive disorder, autism, and schizophrenia
(Treiman 2001; Nordstrom and Burton 2002; Casanova et al.
2003; Lisman et al. 2008). To assess the functional consequen-
ces of bumetanide treatment, we conducted a broad screen for
differences in various fundamental behavioral domains. We
began our analysis in control or bumetanide-treated (E15--P7)
young pups to assess their ability to reach developmental
milestones. On average, bumetanide-treated pups weighed less
than their age-matched controls during the treatment period,
as expected due to this drug’s diuretic effects on the kidney
(Fig. 5A). Bumetanide did not affect the age at which the pups
attained the grasp reflex, surface righting, pinnae detachment,
air righting, and cliff avoidance reflexes (Supplementary
Fig. 3A--E). However, it did delay the pups’ negative geotaxis,
bar holding, and visual placing abilities (logrank test: negative
geotaxis: P <0.05; bar holding: P <0.05; visual placing: P <0.05;
n = 30 animals per condition Fig. 5C--E). Furthermore,
pups treated with bumetanide were less active compared
with controls (Fig. 5B). As a control for reduced weight
from diuresis, we treated mice with high doses of the diuretic
HCTZ from E15 to P7 and assessed their development. As an
additional control for the systemic effects of bumetanide, we
gave daily bumetanide injections to pups from P7 to P14
(bumetanide P7 to P14), during a period when GABA is no
longer depolarizing. HCTZ-treated mice weighed less than
their age-matched controls until P7 when the injections
stopped, and similarly, bumetanide treatment from P7 to P14
decreased the weight of the pups during that period
(Supplementary Fig. 4A). However, neither HCTZ or bumeta-
nide treatment from P7 to P14 resulted in any signs of the
developmental delays seen in bumetanide-treated mice (Sup-
plementary Fig. 4B--J). These observations suggest that reduced
weight due to bumetanide treatment is unlikely to have caused
the behavioral abnormalities seen and that bumetanide
treatment during the perinatal period results in decreased
strength and motor coordination.
Bumetanide Treatment Decreases Anxiety-Related
Behavior and Impairs PPI of the Startle Reflex
We performed a battery of behavioral tests to assess possible
differences between control and bumetanide-treated (E15--
P7) adult mice (8--12 weeks old). We began with general
neurological tests and found no significant difference between
control versus bumetanide-treated animals’ weights, activity
levels, activity intervals,andlatency tostop twisting
Figure 4. Bumetanide treatment decreases spine density of cortical pyramidal neurons. (A) and (B) images of secondary dendrites from 4-week-old cortical pyramidal neurons
from control or bumetanide-treated mice electroporated with GFP at E15. (C) Quantification of the average spine density shows that neurons from bumetanide-treated animals
exhibited a significant spine decrease compared with controls. Scale bar, 20 lm. Bar graphs indicate mean ± SEM (PBS: n 5 56, 3 animals; bumetanide n 5 47, 4 animals;
***P\0.0001, t-test). Quantification of reconstructed cells demonstrated that total dendrite length (D), total dendrite volume (E), total number of dendrite segments (F), number
of dendrite terminal points (G), and full branch level (H) are all significantly decreased in bumetanide-treated pyramidal neurons compared with control. Bar graphs indicate
mean ± SEM (PBS: n 5 17 cells, 3 animals, bumetanide n 5 18 cells, 4 animals, ***P \ 0.0001, t-test).
Cerebral Cortex March 2011, V 21 N 3 581
(Supplementary Fig. 5A). The developmental delays seen in
negative geotaxis and bar holding were not present in
adulthood as both groups performed comparably in these tasks
(Supplementary Fig. 5A). To test for activity and anxiety-related
behavior, we performed the open field test and found that
bumetanide treatment did not change the total movements
made in the center or periphery of the field (Supplementary
Fig. 5B). To test for social behavior, we placed the animals in
the center chamber of a 3-chambered cage. The test occurred
in 3 phases: the habituation phase (right and left chambers
were empty), the socialization phase (empty side and social
side with a bait mouse), and novelty phase (familiar mouse on
one side and novel mouse in the other). In these assays, animals
with autism-like behaviors would spend less time socializing
and have problems recognizing novel objects. However, we did
not see any significant differences between control and
bumetanide-treated animals, though it took a longer time
for bumetanide-treated animals to initiate socialization with
both the familiar and novel mice in the cage (Supplementary
To further test for anxiety-related behaviors, we performed
the elevated plus maze, where a tendency to spend more time
in the open arms is thought to be an operational measure of
decreased anxiety or emotionality (Dawson and Tricklebank
1995). Bumetanide-treated animals showed significantly in-
creased entries into the open arms and trended toward
spending more time and traveling longer distances than
controls in the open arms (PBS vs. bumetanide open entries:
8.1 ± 1.0 vs. 11.4 ± 1.0; n = 30 animals per condition, t-test,
P < 0.05; Fig. 6A). There was no significant difference between
the 2 treatment groups in closed arm time, distance, and
entries (Fig. 6B).
One of the widely used cross-species behavioral paradigms
to measure sensorimotor gating, commonly altered in schizo-
phrenia patients, is the PPI of the startle response (Swerdlow
et al. 2000; Braff et al. 2001). PPI occurs when a low-intensity
prepulse precedes a startle stimulus, resulting in a reduced
startle response. To assess whether bumetanide resulted in
sensory or startle disturbance, we first tested the animals’
maximum startle response. We found that bumetanide-treated
animals showed a significantly higher startle amplitude
compared with PBS-treated controls with the 120-dB stimulus
without a prepulse (PBS vs. bumetanide 120-dB stimulus:
2.32 ± 0.21 N vs. 2.95 ± 0.11 N, n = 29 per condition, t-test,
< 0.05; Fig. 6C). This indicates that bumetanide does
not impair, but rather, may increase the animal’s sensory
Figure 5. Bumetanide treatment causes developmental delay in young mouse pups. (A) Average weights of animals treated with either saline or bumetanide from E15 to P7.
Stars above data point represent those differences that are statistically significant. (B) Count of each motion (no movement, head raise, forelimb raise, or walking) when an
animal is observed for 5 min. Stars in bars represent those movements that are significantly different from those of controls. (C) Percent of animals in each group that are able to
turn around on a negative slope when initially placed head down. (D) Percent of animals able to hang from a bar for longer than 10 s (E) Percent of animals able to raise head and
forelimbs when lowered to a surface by tail. Data points indicate mean ± SEM (n 5 30 animals per condition, *P\0.05, **P\0.001, ***P\0.0001, t-tests for A and B,
logrank tests for C--E).
Bumetanide Alters Cortical Circuits and Sensorimotor Gating Functions
Wang and Kriegstein
reactivity. Importantly, when we analyzed control and bume-
tanide groups for their maximal PPI baseline, we found that PPI
was significantly impaired in mice treated with bumetanide
compared with that of PBS treatment (2-way ANOVA, n = 29
per condition, P < 0.0001; Fig. 6D). Because the differences in
initial startle response might confound the effects of PPI, we
performed a post hoc analysis of animals with similar startle
response. We selected animals that responded to the 120-dB
stimulus with >1.5 N for both control (n = 21) and bumetanide-
treated (n = 29) groups. In our post hoc analysis, the decrease
Figure 6. Bumetanide treatment causes alterations in anxiety-related behavior and sensorimotor gating. (A) Elevated plus maze testing for anxiety-related behavior and
emotionality. Graphs show percent of time spent in the open arms (left), percent of distance traveled in the open arms (middle), and number of entries into the open arms (right).
(B) Same parameters shown for the closed arms. (C) PPI of the startle response testing for sensorimotor gating functions. Different startle amplitudes (in newtons) in response to
the 120-dB stimulus (stim) are graphed against the different prepulse (pp) values. (D) PPI is measured by the degree to which the maximal startle response is inhibited by the
prepulse stimulus. Bar graphs indicate mean ± SEM (n 5 29 animals per condition). (E) Post hoc analysis of mice with similar startle response amplitudes demonstrates that
even in mice matched for maximal startle, bumetanide-treated animals exhibit significant decrease in their PPIs (F) (PBS: n 5 21 animals, bumetanide n 5 29 animals; *P \
0.05, **P \ 0.001, ***P \ 0.0001, t-tests for A and B, 2-way ANOVA for C--F).
Cerebral Cortex March 2011, V 21 N 3 583
in PPI is still significant between the treatment groups despite
having no significant differences in their startle response,
confirming our finding of impaired sensorimotor gating seen
with bumetanide treatment (2-way ANOVA of PPI, P < 0.0001;
Fig. 6E,F). To further ensure that the impairment in PPI is
specific to perinatal bumetanide treatment, we repeated these
tests with another cohort of animals treated with PBS, HCTZ, or
bumetanide from P7--P14. While animals in the bumetanide
P7--P14 group did show a significant increase in their startle
response to the 120-dB stimulus without the prepulse
compared with the PBS-treated controls (PBS vs. bumetanide
P7--P14 120-dB stimulus: 1.27 ± 0.16 N vs. 2.08 ± 0.16 N, n = 12
per condition, t-test, P < 0.05; Supplementary Fig. 6A), neither
HCTZ or bumetanide P7--P14 demonstrated the decreases in
PPI seen in animals treated perinatally with bumetanide (2-way
ANOVA, P > 0.05, Supplementary Fig. 6B). These data suggest
that blocking NKCC1 activity, and thus abolishing the
excitatory effects of GABA, during a critical period results in
functional impairments in sensorimotor gating functions in the
A precise balance of excitatory and inhibitory synaptic inputs is
essential for the proper function of mammalian cortical
circuits. Disruptions in this balance can lead to severe
neurological and psychological disorders. We now provide
novel evidence that systemic blockade of early GABA-mediated
depolarization leads to lasting disruption in AMPA receptor-
mediated glutamatergic transmission in the adult cortex. First,
using electrophysiological recording, we show that abolishing
GABA-mediated excitation in vivo during a critical period
inhibition in the adult cortical circuit. Second, we show through
an array of morphological analyses that dendritic arbors as well
as spine density are reduced when GABA excitation is blocked.
Finally, we show that treating animals with bumetanide during
the perinatal period results in developmental delay and
impairment in sensorimotor gating. These findings support
a cautious approach to the clinical use of bumetanide in infants
since cortical circuits are still maturing at this stage.
A variety of studies have shown the importance of GABA-
mediated depolarization in excitatory synapse development. In
Xenopus tectal neurons, a premature hyperpolarizing shift of
EGABAincreased the ratio of inhibitory to excitatory synapses
(Akerman and Cline 2006). In the mouse neocortex, altering
the chloride gradient of immature cortical neurons in vivo also
disrupted AMPA synapse development and altered the balance
between inhibitory and excitatory synapses (Wang and
Kriegstein 2008). Here, we not only show that this disruption
in excitatory synaptic transmission is long lasting but also that
there is a critical period for excitation--inhibition matching that
occurs around E17--P7 in mice, a period that corresponds to
robust cortical synaptogenesis and overlies the developmental
window when GABA is depolarizing (Owens et al. 1999;
Ben-Ari 2006). Interestingly, blocking GABA-mediated depolar-
ization during either the prenatal period (E15--E19) or post-
natal period (P0--P7) did not produce a persistent effect on
AMPA transmission. This suggests that the window from E17 to
P7 is a sensitive period during which immature cortical
neurons are primed to receive glutamatergic inputs. Any
moderate level of depolarizing activity during this period
would allow the neurons to develop appropriate levels of
AMPAergic inputs. Therefore, only inhibiting NKCC1 activity
during this entire period would be able to produce a lasting
disruption in excitatory synapse development. In addition, the
normal amplitudes and kinetics of the AMPA and GABA mPSCs
suggest that the decrease in excitatory mPSC frequency is
likely due to an overall decrease in the number of functional
excitatory inputs rather than postsynaptic scaling.
The morphological changes in cortical neurons that result
from bumetanide treatment confirm 2 previous reports that
examined the consequences of abolishing the excitatory
effects of GABA in development. These previous studies used
a single-cell genetic approach to alter EGABA and produced
cortical neurons with fewer and shorter dendrites as well as
decreased spine density (Cancedda et al. 2007; Wang and
Kriegstein 2008). The anatomical disruption resulting from
bumetanide treatment is correlated with the physiological
changes seen since dendritic spines constitute major post-
synaptic sites for excitatory synaptic transmission. Our data
seem to contradict the results of a recent study whereby
morphology and synaptic density of hippocampal CA1 pyrami-
dal neurons are unaffected in Nkcc1-null mice (Pfeffer et al.
2009). This discrepancy may be partially attributed to
compensation that occurred in their genetic model as GABA
remains slightly depolarizing in Nkcc1-knockout mice. Our
short-term pharmacological manipulation bypasses this caveat
the hyperpolarization caused by GABA may explain the
morphological changes seen in our study. More interestingly,
these types of morphological abnormalities have been linked to
a variety of neurological and psychiatric disorders including
schizophrenia. For instance, mice with a microdeletion of
22q11.2 or perturbation in the neuregulin-1 signaling cascade,
both of which are highly associated with schizophrenia, show
decreased spine and glutamatergic synapse density, as well as
impaired dendrite growth (Li et al. 2007; Mukai et al. 2008).
We demonstrate that treatment with bumetanide during
a critical early period results in developmental delay and long-
term impairment in sensorimotor gating. Bumetanide-treated
pups demonstrated a delay in achieving milestones associated
with motor coordination and strength (negative geotaxis and
bar holding), and a relative decrease in locomotor activity. Even
though these delays do not persist into adulthood, they mark
disruptions in normal development. These observations suggest
that it may be important to monitor for developmental delay in
clinical trials where bumetanide is used to treat neonatal
Even though bumetanide treatment was systemic, the
altered behavioral phenotypes seen in adult mice are most
likely due to the changes in cortical synaptic transmission. No
evidence suggests that Swiss-Webster mice have any pre-
disposition to neurological or behavioral phenotypes. Because
the brain matures in a caudal--rostral gradient, by E15, NKCC1
expression is largely confined to the telencephalon and, by P0,
largely to the cortical plate (Li et al. 2002). Therefore, our
bumetanide treatment window should primarily affect cortical
neurons without affecting other subcortical structures. Fur-
thermore, bumetanide treatment from P7 to P14 (after EGABA
becomes hyperpolarizing) showed no disturbance in cortical
circuit development or behavioral abnormalities in PPI. PPI is
thought to reflect sensorimotor gating, which is a fundamental
component of information processing in the brain necessary
for stimulus recognition and sequential organization of
Bumetanide Alters Cortical Circuits and Sensorimotor Gating Functions
Wang and Kriegstein
behavior and is severely disrupted in patients with schizophre-
nia due to altered forebrain circuits that regulate inhibition via
descending influences on the pontine tegmentum (Braff et al.
2001; Swerdlow et al. 2001; Bast and Feldon 2003). Interest-
ingly, many lines of evidence have linked glutamate dysfunction
in the cortico-limbic circuit with schizophrenia. For instance,
the noncompetitive NMDA antagonist phencylidine (PCP) can
produce a drug-induced model of schizophrenia with impair-
ments in PPI as well as other positive symptoms such as visual
and auditory hallucination (Jentsch and Roth 1999; Lisman
et al. 2008). Acute treatment of experimental animals with PCP
produces an overall decrease in the level of AMPA glutamate
receptor density especially in the hippocampus, dentate gyrus,
parietal cortex, and amygdala (Zavitsanou et al. 2008). In
addition, neuregulin-1 dysfunction, which has been strongly
associated with schizophrenia, is thought to contribute to the
disease by destabilizing synaptic AMPA receptors leading to loss
of synaptic NMDA currents and reduced number of spines (Li
et al. 2007). Further evidence of involvement of glutamatergic
signaling in schizophrenia is demonstrated in AMPA GluR1
knockout mice, which show PPI impairments and other
cognitive defects seen in the disease (Wiedholz et al. 2008).
In our model, we effectively decreased cortical AMPA synaptic
transmission by blocking GABA-mediated depolarization during
several features that resemble PCP models of schizophrenia,
including PPI impairment, increase in the acoustic startle
reflex, and decrease in AMPA receptor function in cortical
structures (Curzon and Decker 1998; Geyer et al. 2002; Mukai
et al. 2008). While it is unknown if all parts of the cortico-
limbic circuit are affected by bumetanide treatment, future
studies might elucidate the specific alterations in this circuit.
Whether our animal model of bumetanide-treated mice is
a valid model for bumetanide treatment of human neonatal
seizures remains to be elucidated. In rodents, NKCC1 is
expressed from mid-embryonic stage (~E12) until the first
few weeks of postnatal life with the highest expression just
before birth until the first postnatal week (Plotkin et al. 1997; Li
et al. 2002; Wang et al. 2002; Dzhala et al. 2005). Similarly,
Dzhala et al. (2005) demonstrated that in the human cortex,
NKCC1 is expressed from midgestation (~gestational week 20)
until the first year of life with the highest expression occurring
during the perinatal period. We found that the minimal window
to cause deficits in cortical synapse formation is from E17 to P7
in mice, which correlates with the period of high NKCC1
expression. While current clinical trials do not involve
bumetanide treatment during the prenatal period, given the
differences in the length of cortical development between
human and rodents, and our limited ability to test measurable
behaviors in rodents, our results still warrant a cautionary
approach to using bumetanide long-term in treating neonatal
An abnormal balance between excitation and inhibition of
cortical circuits has been implicated in schizophrenia (Lisman
et al. 2008). Recently, a genome-wide screen found rare
structural variants that disrupt multiple genes in schizophrenia,
and pathways involving neuregulin and glutamate were
disproportionately affected (Walsh et al. 2008). Included in
these pathways are genes involved in potassium--chloride
transport, SLC12A9 and SLC12A6, which affect neuronal
excitability (Moser et al. 2008; Walsh et al. 2008). Our data
indicate that transient early bumetanide exposure produces
a persistent clear disturbance in the formation of excitatory
synapses in cortical neurons and leads to deficits in sensori-
motor gating. While our data suggest caution for long-term use
of bumetanide to treat neonatal seizures, side effects in short-
term use remain to be evaluated, and bumetanide may still be
beneficial in treating certain forms of epilepsy in the adult brain
where altered neuronal chloride transport might be involved
(Cohen et al. 2002; Pond et al. 2006).
materialcanbe foundat: http://www.cercor
National Institute of Health to A.R.K.; Medical Scientist Training
Program to D.D.W.; American Epilepsy Society to D.D.W.;
Genentech to D.D.W.
We thank Nino Devidze, Tracy Hamto, Kimberly Scearce-Levie, and
Libby Wilkinson at the Gladstone Neurobehavioral core for performing
the adult animal behavioral tests, members of the Kriegstein laboratory
for discussion and manuscript editing, W. Walantus and J. Agudelo for
technical support. Conflict of Interest: None declared.
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