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Asingle episode of neonatal seizures permanently alters glutamatergic synapses.

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A Single Episode of Neonatal Seizures
Permanently Alters Glutamatergic Synapses
Brandon J. Cornejo, BS,1,2 Michael H. Mesches, PhD,1,3,4 Steven Coultrap, PhD,1
Michael D. Browning, PhD,1,5 and Timothy A. Benke, PhD, MD1,3,5,6
Objective: The contribution of seizures to cognitive changes remains controversial. We tested the hypothesis that a single
episode of neonatal seizures (sNS) on rat postnatal day (P) 7 permanently impairs hippocampal-dependent function in mature
(P60) rats because of long-lasting changes at the synaptic level.
Methods: sNS was induced with subcutaneously injected kainate on P7. Learning, memory, mossy fiber sprouting, spine
density, hippocampal synaptic plasticity, and glutamate receptor expression and subcellular distribution were measured at P60.
Results: sNS selectively impaired working memory in a hippocampal-dependent radial arm water-maze task without inducing
mossy fiber sprouting or altering spine density. sNS impaired CA1 hippocampal long-term potentiation and enhanced long-term
depression. Subcellular fractionation and cross-linking, used to determine whether glutamate receptor trafficking underlies the
alterations of memory and synaptic plasticity, demonstrated that sNS induced a selective reduction in the membrane pool of
glutamate receptor 1 subunits. sNS induced a decrease in the total amount of N-methyl-D-aspartate receptor 2A and an increase
in the primary subsynaptic scaffold, PSD-95.
Interpretation: These molecular consequences are consistent with the alterations in plasticity and memory caused by sNS at the
synaptic level. Our data demonstrate the cognitive impact of sNS and associate memory deficits with specific alterations in
glutamatergic synaptic function.
Ann Neurol 2007;61:411– 426
Approximately 3 in 1,000 infants suffer from neonatal
seizures (seizures occurring in the first month of life);
16% of these children develop learning disabilities
speculatively mediated in part by the seizures themselves.1 Neonatal seizures are often repetitive and prolonged.2 Severe neonatal seizures, or status epilepticus,
have been independently associated with an adverse developmental outcome.3 The cost of educating children
with learning disabilities can be two to five times that
of their peers.4 Treating the impact of neonatal seizures
necessitates an understanding of the mechanisms involved.
Models of neonatal seizures eliminate factors such as
concurrent illnesses, prior brain insult, medication effects, and behavioral interactions that impact learning
ability. In developing rats, multiple episodes (ie, over
several days) of early-life seizures result in later life
learning impairment that is correlated with hippocampal cell loss and synaptic reorganization.5–7 In direct
contrast, immature rats experiencing a single episode
(ie, over a single day) of early-life seizures do not suffer
later-life behavioral alterations, cell loss, or synaptic reorganization in many studies.8 –12 However, recent
work demonstrates that a single episode of early-life
seizure may impair hippocampal-dependent memory
and synaptic plasticity through alterations in inhibitory
synaptic transmission.13
Most excitatory synaptic transmission in the central
nervous system is mediated by glutamate receptorchannels classified according to their preferred agonists:
kainate, ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), or N-methyl-D-aspartate (NMDA).14
Cloned subunit subtypes for AMPA (GluR1-4) and
NMDA receptors (NR1, NR2A-D) have been identified.14 Ca2⫹ influx through NRs is thought to mediate
long-term potentiation and depression (LTP and
LTD), the in vitro assays of rodent learning and memory.15 Postsynaptic changes in GluR subunit numbers16 or properties17 are thought to underlie synaptic
From the Department of 1Pharmacology, 2Medical Scientist Training Program, and 3Department of Pediatrics, University of Colorado, School of Medicine; 4Veterans Affairs Hospital; 5Neuroscience
Program and 6Department of Neurology, University of Colorado,
School of Medicine, Denver, CO.
Published online Feb 23, 2007 in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/ana.21071
Received Jul 15, 2006, and in revised form Nov 6. Accepted for
publication Dec 1, 2006.
Address correspondence to Dr Benke, Departments of Pediatrics,
Neurology, and Pharmacology, University of Colorado, School of
Medicine, Box B-182, 4200 East 9th Avenue, Denver, CO 80262.
E-mail: tim.benke@uchsc.edu
This article includes supplementary materials available via the Internet at http://www.interscience.wiley.com/jpages/0364-5134/suppmat
Published 2007 by Wiley-Liss, Inc., through Wiley Subscription Services
411
modification. Scaffolding proteins such as PSD-95 regulate these changes.18
We expand earlier work investigating the effect of
sNS on inhibitory synapses.13 In this report, we have
associated the effects of a single episode of neonatal
seizures (sNS) induced by kainate acid (KA) at postnatal day 7 (P7) with alterations of hippocampal excitatory synapses at the behavioral, cellular, and molecular
levels. sNS caused permanent alterations in memory after P60. Measurements in vitro demonstrated a permanent adaptation of “plasticity of plasticity” or metaplasticity19 caused by sNS. GluR1 was shifted to
intracellular pools, whereas expression of PSD-95 was
enhanced and NR2A was decreased. We present an excitatory single-synaptic model incorporating our molecular findings and using the subunit “rules” for synaptic
plasticity20 that explains the alterations in plasticity.
Our findings advance the understanding of how neonatal seizures affect memory and glutamatergic, excitatory synaptic function.
Materials and Methods
Animals
All studies conformed to the requirements of the National
Institutes of Health Guide for the Care and Use of Laboratory
Animals and were approved by the Institutional Animal Care
and Use subcommittees of the Denver VA Medical Center
and the University of Colorado Health Sciences Center.
Timed-pregnant Sprague–Dawley rats (Charles Rivers Labs,
Wilmington, MA) gave birth in-house. All rats were housed
in microisolator cages with water and chow available ad libitum.
Seizure Induction
KA, a fixed glutamate analog,14 was used to induce
temporal-lobe seizures.21 After injection, it subsequently
leads to excess glutamate, and thus represents any insult leading to an excess of glutamate including hypoxia.22 Male rat
pups were subcutaneously injected with KA (1–2mg/kg;
Tocris, Ellisville, MO) on P7 (P0 defined as the date of
birth), resulting in discontinuous behavioral and electrical
seizure activity lasting up to 3 hours. P7 was chosen as the
most sensitive time in development to the long-term effects
of sNS,13 and it correlates with human neonatal seizures
clinically,2 biochemically23 and electrographically.24 Onset of
seizure activity occurred within 30 minutes of injection and
was characterized by intermittent myoclonic jerks, generalized tonic-clonic jerks, scratching, “swimming,” and “wetdog shakes.” Mortality was less than 3%. Ictal bursts were
typically less than 10 minutes, separated by 5 to 10 minutes,24 and not consistent with prior definitions of status epilepticus (ictal bursts ⬍ 20 minutes) in this model, which can
be associated with eight times greater mortality.11 In contrast, ictal bursts longer than 20 minutes were observed after
KA only in animals older than P12 (data not shown). Because of discrepancies in the definition of status epilepticus,
which have included ictal discharges as brief as 5 minutes in
adult humans,25 we have not applied this term for clarity.
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Control male rat pups were injected with an equivalent volume of 0.9% saline. Male pups were chosen to eliminate the
effects of hormonal cycles on behavior. Rats were then
tagged using a commercially available microchip tagging system (Avid Identification Systems, Norco, CA) so that experimenters remained blinded to the treatment. Offspring were
returned to their dam after observable seizure activity ceased.
Rats were weaned and separated according to sex at P22. At
P60 to P90 behavioral, histological, electrophysiological, and
biochemical analyses were undertaken with male rats. Four
litters (average 10 pups/litter, 50% male) were used for the
Morris water maze (MWM) and 5 separate litters for the
radial arm water maze (RAWM); one litter was used in both.
Timm’s staining was performed on four litters used for
MWM and RAWM testing. Two separate litters were used
for Golgi staining. Six separate litters were used for electrophysiology. Six separate litters were used for biochemistry.
Morris Water Maze
Rats were trained in the standard hippocampal-dependent
MWM (1.5m diameter, 10cm diameter platform submerged
1cm below the surface of 21°C water obscured with black
tempura paint [Prang, Heathrow, FL], as described previously26). Two blocks of four trials each occurred over 5 days.
During each training trial, the amount of time spent in each
quadrant, distance traveled, and latency to target platform
were measured. If the rat did not find the target platform
within 180 seconds, it was guided there and allowed to stay
for 30 seconds. During the probe trials, the amount of time
spent in each quadrant, distance traveled, latency to target
platform, time spent in the target zone (a smaller region approximately 13cm in diameter around the target location),
and number of platform crossings were recorded. Automated
data collection using WaterMaze (Actimetrics, Evanston, IL)
incorporated a video camera connected to a personal computer.
Four-Trial Radial Arm Water Maze
We used the four-trial radial arm water maze (4T-RAWM)
to confirm MWM findings because it is a more difficult
task27,28 and to train rats for episodic-like memory testing
(see later). The 4T-RAWM27,28 consisted of 12 arms (15cm
wide ⫻ 43cm long) emanating from a circular choice area
(60cm in diameter) in a 1.5m diameter tank of 21°C water.
An escape platform (10 ⫻ 15cm, with a black surface to
match the tank background) was situated at the end of one
of the arms, approximately 2cm below the surface of opaque
water. Rats were pretrained in the maze for 5 days. Pretraining consisted of shaping the rats to find the target arm by
initially preventing entry into the nontarget arms and gradually increasing the number of available arms until all 12
were open. Testing began on day 6, and the rats were trained
over 4 days. The start arm for each trial was determined in a
pseudorandom fashion with a given arm used once per day.
The start and goal arms were different for each rat within a
group on a given day, but equivocal across groups, to avoid
place and position preferences. The goal arm for a given rat
remained the same each day. Four trials were administered
per day (maximum, 180 seconds) with a 30-second intertrial
interval. If the rat did not find the escape platform within
180 seconds, it was guided to the correct arm and allowed to
stay for 30 seconds.
Two-Trial Radial Arm Water Maze with Delay
We used the two-trial RAWM with delay (2T-RAWM) to
isolate episodic-like memory deficits that can be seen with
normal performance in the MWM and are critically dependent on hippocampal NMDA receptors.29 Testing in the
2T-RAWM followed the 4T-RAWM on day 14 over 4 days.
One training trial was administered (maximum, 180 seconds). If the rat did not find the target platform within 180
seconds, it was guided to the correct arm and allowed to stay
for 30 seconds. A 4-hour delay was inserted after the training
trial. In the delay trial, the target platform was in the same
location as the training trial for a given rat. The start location was novel for each rat during the delay trial. If the rat
did not find the target platform within 180 seconds, it was
guided to the correct arm and allowed to stay for 30 seconds.
During RAWM trials, each rat was assessed for total errors
(ie, entry into an arm that did not contain the escape platform), latency to the target platform, and repeat errors (reentry into an arm previously visited). Data were collected in
a similar fashion to the MWM. Data are presented as the
mean ⫾ standard error of the mean for each trial across 4
days.
The results for each rat were pooled from each scorer and
region, averaged, and reported as a Timm’s staining score for
both the DG and CA3 regions. Slides with poor staining or
artifact were not scored.
Golgi Staining
After anesthesia with an injection of pentobarbital
(120mg/kg intraperitoneally), rats were transcardially perfused with 0.1M phosphate buffer (pH 7.4) followed by
10% formalin in 0.1M phosphate buffer. The brain was rapidly removed and rinsed in 0.1M phosphate buffer. The FD
Neurotechnologies Rapid Golgi Stain Kit (FD Neurotechnologies, Ellicot City, MD) was used according to the manufacturer’s proprietary directions. Coronal sections (90␮m)
were made through the hippocampus using a sliding microtome and mounted onto gelatin- and chrom alum–coated
slides and stained. Sections were coverslipped using DPX
mountant (BioChemika). The CA1 stratum radiatum region
of representative sections was visualized at 100 to 1,000
times and scored for spine density (density calculated as the
number of spines/total length of dendrite) and branching
number along the primary apical dendrite of pyramidalshaped neurons.32 Typically, 391 ⫾ 10␮m/dendrite was
scored (N ⫽ 123 neurons; range, 146 – 693␮m). Slides with
poor staining or artifact were not scored.
Timm’s Staining
Hippocampal Slice Preparation and Electrophysiology
Timm’s staining was performed as described previously.30
After anesthesia with an injection of pentobarbital
(120mg/kg intraperitoneally), rats were transcardially perfused with a sodium sulfide solution followed by 10% formalin in 0.1M phosphate buffer. Brains were removed, cryoprotected for 2 days in 10% formalin with 20% glycerol in
0.1M phosphate buffer, then rapidly frozen on dry ice/acetone. Coronal sections (30␮m) were made through the hippocampus using a sliding microtome and mounted onto
gelatin- (Fisher, Hampton, NH) and chrom alum–coated
(Fisher) slides. In the dark, mounted sections were placed in
a solution of gum arabic, citrate buffer, and hydroquinone
for 30 to 45 minutes or until the molecular layer in the
dentate gyrus (DG) was clearly stained. Sections were defatted, counterstained with cresyl violet (Standard Fluka, St.
Louis, MO), and coverslipped with DPX mountant (BioChemika, St. Louis, MO). Scoring by three separate individuals at a total magnification of 400 to 1,000 times was performed as described elsewhere31: The CA3 and DG regions
were scored on a scale of 0 (no granules in the supragranular
region of DG and no granules in the stratum pyramidal or
stratum oriens along any portion of the CA3 subregion), 1
(occasional granules in the stratum pyramidal or stratum
oriens occurring in discrete bundles), 2 (occasional to moderate granules in the stratum pyramidal or stratum oriens), 3
(prominent but discontinuous granules in the stratum pyramidal or stratum oriens), 4 (prominent granules in the stratum pyramidal or stratum oriens occurring in nearcontinuous distribution along the entire CA3 region), or 5
(continuous or near-continuous dense laminar band of granules in the supragranular region of DG and continuous or
near-continuous dense laminar band of granules stratum pyramidal and stratum oriens along the entire CA3 region).
After rapid decapitation and removal of the brain, sagittal
hippocampal slices (400␮m) were made using a vibratome
(Vibratome, St. Louis, MO) in ice-cold sucrose artificial cerebrospinal fluid (saCSF; 206mM sucrose, 2.8mM KCl,
1mM CaCl2, 3mM MgSO4, 1.25mM NaH2PO4, 26mM
NaHCO3, 10mM D-glucose and bubbled with 95%/5% O2/
CO2).33 Slices were recovered in a submersion-type chamber
perfused with oxygenated artificial cerebrospinal fluid (aCSF;
124mM NaCl, 3mM KCl, 1mM MgSO4, 2mM CaCl2,
1.2mM NaH2PO4, 26mM NaHCO3, 10mM D-glucose and
bubbled with 95%/5% O2/CO2) at room temperature for at
least 60 minutes, and then submerged in a recording chamber perfused with aCSF. All electrophysiology was performed
in the CA1 region. Two twisted-tungsten bipolar stimulating
electrodes were offset in the CA1 to stimulate the two independent Schaffer collateral-commissural pathways using a
constant current source (WPI, Sarasota, FL) with a fixed duration (20 microseconds), each at a rate of 0.033Hz. Field
excitatory postsynaptic potentials (fEPSPs) were recorded
from the stratum radiatum region of CA1 using a borosilicate glass (WPI) microelectrode (pulled to 6 to 9M⍀ when
filled with 3M NaCl; Sutter, Novato, CA,), amplified 1,000
times (WPI and Warner, Hamden, CT), and digitized (CIODAS08/JR-A0; Measurement Computing, Middleboro, MA)
at 10kHz using LTP-version 2.4.34 Input-output curves, as a
measure of basal synaptic function, were generated at four
stimulus intensities: 10, 20, 50, and 95% of maximal slope
value (in msec/mV). Paired-pulse facilitation, an index of
functional presynaptic CA1 glutamate release, was measured
by two pulses 50 milliseconds apart. Paired-pulse facilitation
associated with presynaptic function is maximal at this interpulse interval.35 Input-output curves and paired-pulse facilitation were measured after obtaining a stable baseline of 30
Cornejo et al: Neonatal Seizures Alter Synapses
413
minutes. After baseline stabilization of fEPSP slope at approximately 50% of maximal slope for at least 20 minutes,
LTP was induced (100Hz ⫻ 1 second) in one Schaffer
collateral-commissural pathway, whereas LTD was induced
60 minutes after LTP in the other pathway (900 paired-pulse
stimuli at 1Hz with 50-millisecond interpulse interval). Slices
with unstable baseline were discarded.
Subcellular Fractionation
After rapid decapitation, brains were removed and placed in
ice-cold aCSF. The hippocampus was unrolled by inserting a
thin instrument into the hippocampal fissure separating CA1
from the DG. The DG and CA3 regions were then rolled
back and the connection between CA1 and CA3 was cut,
removing CA3 and DG. The subiculum was then cut away
from CA1. From the isolated CA1, 400␮m slices were prepared with a McIlwain tissue chopper. CA1 minislices were
visually inspected to ensure that the other regions had been
more than 99% effectively removed. This method has been
found to produce viable slices.36,37 Homogenization (in
320mM sucrose, 10mM tris[hydroxymethyl]aminomethane
[Tris], pH 7.4) was followed by subcellular fractionation
with slight modification.38 Homogenates were centrifuged at
100 g to remove nuclei and large debris (P1). Crude synaptosomal membranes (P2) were then prepared from supernatants (S1) by centrifugation at 10,000 g. Resulting supernatants (S2) were spun at 100,000 g to obtain the light
membrane fractions (P3). Final pellets were resuspended in
STE buffer (10% sodium dodecyl sulfate, 10mM EDTA,
100mM Tris, pH 8) and boiled for 5 minutes. Protein concentrations were determined using a BCA protein assay kit
(Pierce, Rockford, IL) with BSA (bovine serum albumin) as a
standard. Samples were diluted to equal protein concentrations in sample loading buffer (2.3% sodium dodecyl sulfate,
67.5mM Tris, 10% glycerol, 5% ␤-mercaptoethanol,
0.017% bromophenol blue) and assayed by semiquantitative
Western blot.
Cross-linking
Hippocampal slices that were prepared and recovered as for
electrophysiology were placed into either ice-cold aCSF (to
measure total protein concentration) or ice-cold aCSF containing 1mg/ml BS3 (BIS-[sulfosuccinimidyl] Suberate;
Pierce) (to measure intracellular protein concentration) for
40 minutes at 4°C. To quench remaining BS,3 slices were
washed three times in cold aCSF containing 20mM Tris pH
7.6. Slices were suspended in STE buffer, sonicated, and
samples prepared for Western blotting as with subcellular
fractionation.
Semiquantitative Western Blotting
Western blotting was performed as described previously.39
Samples were loaded in duplicate on 7.5% polyacrylamide
gels and a six-point dilution series of rat hippocampal homogenate was included on each gel as a standard curve.
Blots, after blocking with either 5% Carnation nonfat dry
milk or 3% BSA (Sigma, St. Louis, MO) in TTBS (140mM
NaCl, 20mM Tris, pH 7.6, 0.1% Tween 20 [Sigma]), were
probed overnight with primary antibody at 4°C. Primary antibodies to GluR1 and GluR2/3 (Chemicon, Temecula, CA)
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were diluted 1:3,000 in 1% Carnation nonfat dry milk, NR1
(BD Biosciences, San Diego, CA) was diluted 1:3,000 in 1%
BSA, NR2A and NR2B40 were diluted 1:1,000 and 1:3,000,
respectively in 1% BSA, and PSD-95 (Affinity Bioreagents,
Golden, CO) was diluted 1:5,000 in 1% BSA. Blots were
washed three times with TTBS, probed for 1 hour at room
temperature with horseradish peroxidase–conjugated goat
anti–mouse or goat anti–rabbit secondary antibodies (BioRad, Temecula, CA), then washed three more times with
TTBS. Immunodetection was effected using a chemiluminescent substrate (SuperSignal West Femto Maximum Sensitivity Substrate; Pierce) and an Alpha Innotech imaging system;
digital images were quantified using AlphaEase software (Alpha Innotech, San Leandro, CA). Immunoreactivity was reported as the density of sample bands relative to the standard
curve. Only values falling within the standard curve generated from the dilution series included on each gel were incorporated into the final analysis.
Statistics
Data are expressed as mean ⫾ standard error of the mean,
and n ⫽ number of rats for a given treatment. Two-way
repeated-measures analyses of variance (ANOVAs) with
Holm–Sidak post hoc analysis, two-way ANOVAs, Mann–
Whitney rank-sum, and Student’s t tests were used, where
indicated, for statistical comparisons for electrophysiological,
anatomical, and biochemical data using Origin 7.5 S5R (Origin Lab Corporation, Northampton, MA) or SigmaStat
(Systat, Point Richmond, CA). Significance was reported at
p ⬍ 0.05.
Results
Morris Water Maze and Four-Trial Radial Arm
Water Maze Showed No Effect of Single Episode of
Neonatal Seizures on Spatial Learning and Recall
Previous work has described the utility of the MWM
in assessing hippocampal-dependent spatial learning.26,41 In developing rats, multiple episodes of seizures cause hippocampal-dependent spatial learning
deficits as measured with the MWM.5–7 Recent work
has demonstrated that a single episode of early-life seizures has a negative impact on behavior and
hippocampal-dependent spatial learning using a fooddependent radial arm maze and other behavioral
tests.13 Radial arm mazes require a greater memory
load, and thus are capable of potentially demonstrating
subtle learning and memory deficits.27,28 Our utilization of the RAWM omits the necessity of a food reward and is therefore hippocampal dependent28 and
more comparable with the MWM.
We initially used the MWM in adult rats to assess
potential deficits induced by sNS to directly compare
our results with prior studies. With respect to distance
traveled, two-way repeated-measures ANOVA demonstrated no overall effect of sNS. There was a nonsignificant difference in block 3 (the first block of day 2)
in which control rats traveled less distance to find the
platform than sNS rats (Fig 1A). Rats performed
equally well during the probe trial, indicating that
hippocampal-dependent spatial recall after learning was
grossly undisturbed after sNS (see Fig 1B). Although
no overall effect of training or treatment was observed
in the 4T-RAWM, a pairwise Holm–Sidak analysis following two-way repeated-measures ANOVA showed
that sNS rats (13.15 ⫾ 0.78 total errors; n ⫽ 12)
made significantly more total errors on trial 1 compared with control rats (11.32 ⫾ 0.65 total errors; n ⫽
8) ( p ⫽ 0.004), suggesting a subtle defect in learning.
However, both sNS and control rats acquired the task
equally (see Fig 1C). There were no differences in
swim speed after sNS (25.4 ⫾ 0.5cm/sec; n ⫽ 10)
compared with the control group (25.7 ⫾ 0.5cm/sec;
n ⫽ 11; Student’s t test, p ⫽ 0.68).
Fig 1. Behavioral effects of a single episode of neonatal seizures (sNS) in adult rats were isolated to a deficit in episodic-like or
working memory in the two-trial radial arm water maze (2T-RAWM). (A) Adult male rats that experienced sNS (solid circles) or
saline-injected control rats (open circles; treated on postnatal day 7 [P7] and matured to P60) were trained on the Morris water
maze (MWM). Both sNS (n ⫽ 11) and control rats (n ⫽ 10) performed equally across all days of training regarding distance
traveled. No differences were observed with respect to swim speed (see Results). (B) No differences in percentage time spent in each
quadrant (NE, Target, SW, SE) were detected during the probe trial. (C) In the four-trial (4T)-RAWM, total errors were defined
as an incorrect arm entry and were used to measure the extent of task acquisition and recall. The task was learned equally by both
sNS (n ⫽ 12) and control rats (n ⫽ 8) because both had similar total errors for trials 1 through 4 (T1-T4); however, a pairwise
Holm–Sidak analysis after two-way repeated-measures analysis of variance (ANOVA) demonstrated that sNS rats made slightly but
significantly more total errors on T1 compared with control rats (see Results). (D) For testing in the 2T-RAWM, a 4-hour delay
was inserted between the training trial and the delay trial over the following 4 days (see Methods). After a 4-hour delay, sNS rats
(solid bars) demonstrated a significant increase in the number of total errors made compared with control rats (open bars; 6.58 ⫾
0.75 vs 3.73 ⫾ 0.44) (see Results). (E) Latency to target platform also was used to characterize working memory deficits. sNS rats
had a significantly longer latency to platform during the delay trial compared with control rats (52.38 ⫾ 7.34 vs 29.82 ⫾ 2.97)
(see Results). (F) Repeat errors, a measure of working memory, were defined as an entry into an arm that had been previously visited. sNS rats made significantly more repeat errors than control rats (2.58 ⫾ 0.42 vs 1.02 ⫾ 0.21). Asterisk indicates statistically
significant difference by two-way repeated-measures ANOVA, p ⱕ 0.05.
Cornejo et al: Neonatal Seizures Alter Synapses
415
Two-Trial Radial Arm Water Maze with Delay
Showed Specific Effect of Single Episode of Neonatal
Seizures on Memory
Recent studies suggest that the hippocampus underlies
the “automatic recording” of episodic-like memory in
humans and in rodents.29 Episodic-like memory has
been defined as recall for a single event that involves
spatial, nonspatial, and temporal cues.29 Isolated,
episodic-like memory deficits can be seen with normal
performance in the MWM and are critically dependent
on NMDA receptors.29
The 2T-RAWM directly tested the effects of sNS on
memory. Adult sNS rats made more (6.58 ⫾ 0.75;
n ⫽ 28) total errors than control rats (3.73 ⫾ 0.44;
n ⫽ 29) during the delay trial (see Fig 1D; two-way
repeated-measures ANOVA, F (1, 55) ⫽ 7.928; p ⫽
0.007). sNS rats took longer (52.38 ⫾ 7.34 seconds;
n ⫽ 29) during the delay trial to find the platform
than littermate control rats (29.82 ⫾ 2.97 seconds;
n ⫽ 28) (see Fig 1E; two-way repeated-measures
ANOVA, F (1, 55) ⫽ 4.874; p ⫽ 0.031). The number
of repeat errors (defined as reentries into arms that
were already visited) was also examined. These errors
are considered a measure of working memory and reflect impairment in the mechanisms responsible for
maintaining short-term information processing.29 sNS
rats demonstrated more than twice as many repeat errors (2.58 ⫾ 0.42; n ⫽ 29) during the delay trial compared with control rats (1.02 ⫾ 0.21; n ⫽ 28) (see Fig
1F; two-way repeated-measures ANOVA, F (1, 55) ⫽
6.708; p ⬍ 0.001). Thus, although learning (and recall
after learning) appears grossly normal, this demonstrates a specific alteration of episodic-like or working
memory, or both.
A Single Episode of Neonatal Seizures Did Not
Cause Mossy Fiber Sprouting
We next examined two aspects of hippocampal morphology that have been associated with deficits in
learning and memory. Aberrant mossy fiber sprouting
after multiple episodes of early-life seizures31,42 and
early-life stress43 has been linked with poor performance on the MWM. We found no aberrant mossy
fiber sprouting after sNS (see Supplementary Figs 1A1
and 1A2) (CA3: Mann–Whitney rank-sum, p ⫽ 0.230,
n ⫽ 19 control rats, n ⫽ 18 sNS rats; DG: Mann–
Whitney rank-sum, p ⫽ 0.681, n ⫽ 18 control rats,
s ⫽ 18 sNS rats). As a positive control, seizures were
induced in adult rats by injection with KA (10mg/kg)
on P42. Based on previous data, it was expected that
these rats would have aberrant mossy fiber sprouting in
the hippocampus compared with saline-injected control
rats.31 In confirmation of this, rats that seized as adults
(n ⫽ 2) developed easily detectable mossy fiber sprouting in the DG and CA3 region of the hippocampus
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compared with control rats (n ⫽ 2) (see Supplementary Figs 1B1 and 1B2).
A Single Episode of Neonatal Seizures Did Not Alter
Spine Density or Dendritic Branching
Reduction in spine density after multiple early-life seizures has been linked with poor performance on the
MWM32,44 and in other rodent models of learning and
memory deficits.45 Reduction in spine density is also
found in humans with Down’s syndrome and other
metabolic disorders.46 We examined spine density and
branching by bright-field imaging of Golgi-stained
CA1 hippocampal neurons. There were no changes between sNS (n ⫽ 5; 52 neurons) and control rats (n ⫽
7; 71 neurons) for either spine density (Figs 2A, B) or
branching along primary apical dendrites (see Figs
2A, C).
A Single Episode of Neonatal Seizures Altered
Hippocampal CA1 Plasticity
The lack of evidence for histological changes that could
underlie the sNS-induced alterations in memory led us
to examine the possibility that sNS might alter CA1
hippocampal function or synaptic plasticity in vitro.
We found no significant differences between groups for
paired-pulse facilitation (Fig 3A), suggesting sNS did
not alter presynaptic function. By comparing presynaptic fiber volley size with fEPSP slope at different stimulus intensities (see Fig 3B), we found no significant
difference between groups, suggesting that sNS did not
change overall excitability. LTP and LTD are thought
to reflect the processes involved in learning and memory,15 and consideration of the balance between the
two has been referred to as metaplasticity.19 When a
100Hz ⫻ 1-second stimulus was used to induce LTP,
slices from sNS rats demonstrated a significant and
profound loss of LTP compared with control rats as
measured by percentage change in fEPSP slope (Fig
4A, asterisk indicates p ⱕ 0.05 by Student’s t test). We
next induced LTD using a robust47 stimulus protocol
(900 paired pulses at 1Hz, 50-millisecond interpulse
interval). There was a significant enhancement of LTD
in slices from rats that experienced sNS (see Fig 4B,
asterisk indicates p ⱕ 0.05 by Student’s t test). Thus,
sNS could have been a metaplastic event that altered
the balance between LTP and LTD.
Effects of Single Episodes of Neonatal Seizures on
Glutamate Receptors and PSD-95
Alterations in membrane expression of glutamate receptors,15 either caused by increases or decreases in total numbers, or shifts between membrane and intracellular pools, could play key roles in determining the “set
point” for LTP versus LTD, that is, metaplasticity. To
test the effects of sNS on membrane expression of glutamate receptors, we used both subcellular fraction-
Fig 2. Memory deficits caused by a single episode of neonatal seizures (sNS) were not associated with changes in spine density or
spine branching measured in adult rats. (A) No observable differences in branching pattern or spine density were seen after Golgi
staining (see Materials and Methods) at low (100⫻, left panels; scale bar ⫽ 20␮m) and higher magnification (1,000⫻, right
panels; scale bar ⫽ 10␮m) after sNS (n ⫽ 5; 51 neurons) or for saline control rats (n ⫽ 7; 72 neurons) after postnatal day
(P60). Quantification (B) showed no differences for spine density. Spine densities were calculated as the number of spines per micron along the apical dendrite. Quantification (C) showed no differences for the number of branches on the apical dendrite. The
total number of branches on the selected apical dendrite used for spine density was counted.
ation and cross-linking techniques to separate and measure the concentration of glutamate receptor subunits
in both the membrane and intracellular compartments
of neurons in the CA1 region of the hippocampus. In
the first set of experiments, we prepared both intracellular and surface membrane fractions using classical
subcellular fractionation from control and sNS tissue,
and then probed the fractions with antibodies against
GluR1, GluR2/3, and NR1. We found that there was
a significant (ie, 52%) increase in intracellular GluR1
in CA1 slices prepared from rats that experienced sNS
(2.35 ⫾ 0.26 immunoreactivity/␮g protein; n ⫽ 11)
compared with control rats (1.54 ⫾ 0.21 immunoreactivity/␮g protein; n ⫽ 8) (Student’s t test, p ⫽ 0.03)
(Figs 5A, D). This effect appeared to be specific for
GluR1, because we saw no effect of sNS on levels of
GluR2/3 or NR1 in either the membrane fraction or
intracellular fraction (see Figs 5A–C).
To be certain that the change in GluR1 levels was
not simply an artifact of the subcellular fractionation
assay, we confirmed our results using a cross-linking
methodology. This technique incorporated the use of
the membrane-impermeable cross-linker BS3 that specifically cross-links extracellular domains of membrane
proteins and allows their subsequent separation from
intracellular proteins through gel electrophoresis. The
concentration of total protein is compared with the
measured concentration of intracellular protein and expressed as a percentage of the total. While using this
assay, we detected no change in the total amount of
GluR1. However, we detected a significant increase in
intracellular GluR1 in rats that experienced sNS (n ⫽
8) compared with control rats (n ⫽ 6), as the intracellular percentage increased from 5.8 ⫾ 0.5 to 10.8 ⫾
1.1% (Fig 6A) (Student’s t test, p ⱕ 0.0008). This
percentage change (100*[10.8 ⫺ 5.8]/5.8 ⫽ 85%) was
comparable with that seen with fractionation techniques (52%) given the differences in methodology.
We speculate that a comparable change in the membrane fraction would be difficult to detect because
most GluR1 (100 –5.8% ⫽ 94.2% in control rats to
100 –10.2% ⫽ 89.2% after sNS) is on the membrane
surface. Such a percentage change (100*[94.2 ⫺ 89.2]/
94.2 ⫽ 5.3%) was within the standard error for the
Cornejo et al: Neonatal Seizures Alter Synapses
417
Fig 3. A single episode of neonatal seizures (sNS) did not alter synaptic excitability measured in the CA1 region of adult hippocampal slices. Field excitatory postsynaptic potentials (fEPSPs) were measured from the stratum radiatum of the CA1 region of adult
hippocampal slices in response to stimulation of the Schaffer collateral-commisural pathway (see Materials and Methods). (A) sNS
(n ⫽ 11) did not significantly alter paired-pulse (50-millisecond interpulse interval) facilitation compared with saline-injected control rats (n ⫽ 15). No differences were observed between groups. P1 ⫽ normalized fEPSP slope on first pulse; P2 ⫽ normalized
fEPSP slope after 50-millisecond interpulse interval. (B) Fiber volley amplitude and fEPSP slope were measured at four different
stimulus intensities (see Materials and Methods). Data points for each measurement (averaged from 16 sweeps) from sNS (solid
circles; n ⫽ 8) and saline-injected control rats (open circles; n ⫽ 9) are plotted. The slope of the regression line for sNS (dotted)
and control rats (solid) were similar. Sample traces at 10, 20, 50, and 95% of maximal slope value are shown from control
(thick line) and sNS animals.
measurement of GluR1 in the membrane fraction determined by subcellular fractionation (see Fig 5D). As
shown previously,37 the specificity of BS3 for extracellular domains was confirmed by the absence of any effect of the cross-linker on the intracellular protein synapsin (data not shown). Control animals demonstrated
expected expression patterns for mature animals.48 We
found no differences in either totals or localization of
GluR2/3 or NR1 between control rats and rats that
experienced sNS (see Figs 6B, C), as with subcellular
fractionation. We detected a significant 28% loss of total NR2A in rats that experienced sNS (0.534 ⫾ 0.042
immunoreactivity/␮g protein; n ⫽ 9) compared with
control rats (0.745 ⫾ 0.097 immunoreactivity/␮g pro-
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tein; n ⫽ 9) (Student’s t test, p ⬍ 0.03) (see Fig 6D).
However, the percentage of this total located intracellularly was unchanged by sNS. We found no differences in either totals or localization of NR2B between
control rats and rats that had experienced sNS (see Fig
6E).
PSD-95, one of the most abundant scaffolding proteins, directly interacts with NRs and indirectly with
GluRs.49 PSD-95 is almost completely expressed in the
intracellular compartment of only excitatory synapses.50 Therefore, we used immunoblotting to investigate the intracellular concentration of PSD-95 (Figs
7A, B). We observed a significant 43% increase in the
total concentration of PSD-95 in rats that experienced
Fig 4. A single episode of neonatal seizures (sNS) caused a metaplastic shift that impaired long-term potentiation (LTP) and enhanced long-term depression (LTD) measured in the CA1 region of adult hippocampal slices. Field excitatory postsynaptic potentials
(fEPSPs) were measured from the stratum radiatum of the CA1 region of adult hippocampal slices in response to stimulation of the
Schaffer collateral-commisural pathway (see Materials and Methods). (A) After LTP induction (100Hz ⫻ 1 second), sNS (filled
circles; n ⫽ 10) caused a near-total loss of LTP compared with controls (solid circles; n ⫽ 14). *p ⱕ 0.05, Student’s t test.
Sample averaged sweeps (arrowhead at stimulus artifact, average of four sweeps) show pre- (thin lines) and post-LTP (thick lines)
traces. (B) After an extended baseline period demonstrating stability, sNS rats (n ⫽ 11) displayed significantly enhanced LTD
(900 ⫻ 50-millisecond paired pulses at 1Hz) compared with their littermate controls (n ⫽ 15). *p ⱕ 0.05, Student’s t test. Sample averaged sweeps show pre- (thin lines) and post-LTD (thick lines) traces.
sNS (1.084 ⫾ 0.127 immunoreactivity/␮g protein;
n ⫽ 8) compared with control rats (0.757 ⫾ 0.076
immunoreactivity/␮g protein; n ⫽ 11; Student’s t test,
p ⱕ 0.03). Given that the number of synapses did not
appear to be altered by sNS (see Fig 2), this suggests
that the amount of PSD-95 in each excitatory synapse
was increased after sNS.
Discussion
Our results establish using multiple approaches that
sNS disrupted hippocampal-dependent memory and
plasticity that were correlated with alterations at excitatory, glutamatergic synapses. Although much early
data argued that immature rodents tend to be resistant
to a single episode of seizure-induced changes in
physiology, behavior, or histology (reviewed in
Stafstrom51), recent work13 has challenged this and has
demonstrated altered inhibitory synaptic function.
Now, by linking crucial glutamatergic receptor and
scaffolding changes with plasticity and behavior, we are
mechanistically beginning to understand the harmful
issues underlying just a single episode of relatively mild
neonatal seizures.
Standard behavioral testing (MWM) and a more difficult test (4T-RAWM) determined hippocampaldependent learning and recall were grossly normal in
adult rats after sNS. The 2T-RAWM demonstrated
that sNS specifically impaired episodic-like or working
memory, or both. This mixture of normal learning and
recall with an episodic-like memory deficit is also seen
Cornejo et al: Neonatal Seizures Alter Synapses
419
Fig 5. A single episode of neonatal seizures (sNS) caused an isolated, long-term increase in intracellular glutamate receptor 1
(GluR1) without changes in GluR2/3 or N-methyl-D-aspartate (NMDA) receptor 1 (NR1) localization in adult CA1 hippocampus.
Membrane and intracellular fractions were generated using subcellular fractionation and analyzed on Western blots using a known
standard curve to generate a relative concentration (immunoreactivity/␮g protein) (see Materials and Methods). (A) Example blots
of GluR1, GluR2/3, and NR1. Greater density is observed in the intracellular fraction stained by GluR1 after sNS compared with
control; all other blots are nearly equal. (B) There was no effect of sNS on NR1 concentrations in the membrane (sNS: n ⫽ 9;
control: n ⫽ 8) or intracellular pools (sNS: n ⫽ 8; control: n ⫽ 7). (C) There was no effect of sNS on GluR2/3 concentrations in
the membrane (sNS: n ⫽ 8; control: n ⫽ 9) or intracellular pools (sNS: n ⫽ 7; control: n ⫽ 7). (D) sNS rats resulted in an
increase in intracellular GluR1 concentrations (2.35 ⫾ 0.26 immunoreactivity/␮g protein; n ⫽ 11) compared with their littermate
control rats (1.54 ⫾ 0.21 immunoreactivity/␮g protein; n ⫽ 8; p ⫽ 0.03, Student’s t test;). There was no effect of sNS on
GluR1 concentrations in the membrane (sNS: 1.54 ⫾ 0.14 immunoreactivity/␮g protein; n ⫽ 12; control: 1.54 ⫾ 0.15 immunoreactivity/␮g protein; n ⫽ 10).
in GluR1 knock-out mice52 and in mice in which specific GluR1-modulating phosphorylation sites have
been altered.53 In these knock-out studies, both LTP
and LTD were obliterated. In contrast, we found that
there was an overall shift in plasticity that favored the
expression of greater LTD at the expense of a total loss
of LTP; thus, other plasticity mechanisms emerged
here, as in prior knock-out studies, to sustain MWM
performance. These findings were specifically associated with an increase in the intracellular pool of
GluR1, a net loss of NR2A, and an increase in PSD95. We did not find any changes in GluR2/3, NR1, or
NR2B regarding their total amounts or their distribution. Prior studies by others8,13,54 have not reported
any evidence of cell loss caused by sNS. Therefore, because sNS did not change overall excitability or spine
numbers, we conclude that sNS caused alterations at
the level of individual excitatory, glutamatergic synapses.
The underlying derangements in GluR1, NR2A, and
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PSD-95 that result in altered plasticity at an excitatory
synapse can be explained by saturated or impaired receptor expression, or both, and altered induction
mechanisms (Fig 8). Given the dramatic increase in
PSD-95, the stability in spine numbers, and the use of
field recording techniques that sample many synapses
at a time, we speculate that the majority of synapses in
the CA1 region of the hippocampus have been affected.
Scaffolding proteins such as PSD-95 bring GluRs to
synapses and upregulate them in LTP55,56 and remove
them in LTD.55,57 In cultured neurons, overexpression
of PSD-95 is associated with sustained synaptic enhancement,58 occlusion of LTP,59,60 and enhanced
LTD.59 We speculate that the 43% increase in PSD-95
expression that we observed in adult synapses after sNS
drives a sustained synaptic enhancement or saturation
and partially underlies the profound loss of LTP.
Therefore, it is consistent with prior findings that we
Fig 6. A single episode of neonatal seizures (sNS) caused a long-term shift favoring intracellular glutamate receptor 1 (GluR1) and
a loss of total N-methyl-D-aspartate (NMDA) receptor 2A (NR2A) in adult CA1 hippocampus. Total hippocampal CA1 protein
concentration (immunoreactivity/␮g protein [Imm/␮g protein]) and intracellular fractions (percentage expression [% Intra Exp])
were generated using cross-linking techniques (see Materials and Methods). (A) There was no effect of sNS on total GluR1 concentration (sNS: 0.52 ⫾ 0.05 immunoreactivity/␮g protein, n ⫽ 11; control: 0.57 ⫾ 0.07 immunoreactivity/␮g protein, n ⫽ 8).
There was a significant increase in the intracellular fraction of GluR1 after sNS (10.83 ⫾ 1.1%; n ⫽ 8) compared with saline
controls (C; 5.84 ⫾ 0.54%; n ⫽ 6; p ⫽ 0.008, Student’s t test). (B) There was no effect of sNS on total GluR2/3 concentration
(sNS: n ⫽ 8; control: n ⫽ 9) or intracellular pools (sNS: n ⫽ 8; control: n ⫽ 6). (C) There was no effect of sNS on total NR1
concentration (sNS: n ⫽ 9; control: n ⫽ 8) or intracellular pools (sNS: n ⫽ 9; control: n ⫽ 9). (D) sNS caused a decrease
(0.534 ⫾ 0.042 immunoreactivity/␮g protein; n ⫽ 9) in total NR2A concentration compared with saline controls (0.745 ⫾
0.097 immunoreactivity/␮g protein; n ⫽ 9; p ⫽ 0.03, Student’s t test). However, there were no differences in NR2A concentrations in intracellular fractions between sNS (n ⫽8) and control rats (n ⫽ 8). (E) There was no effect of sNS on total NR2B concentration (sNS: n ⫽ 8; control: n ⫽ 10) or intracellular pools (sNS: n ⫽ 8; control: n ⫽ 10).
observed complete occlusion of LTP and greater LTD
in adult rats that experienced sNS.
After sNS, adult synapses likely have decreased
membrane expression of GluR1, because of a 52 to
85% increase in the amount of GluR1 in the intracellular pool. GluR1 and GluR2/3 are the predominant
GluRs in adult synapses.61 We predict that if there are
fewer total functional GluR receptors after sNS, overall
excitability has to be balanced. This is supported by
our findings of unchanged excitability (see Fig 3). For
this to occur, some individual GluRs must be potentiated or enhanced. Limitations with the biochemical assays used do not permit a differentiation between those
membrane-bound receptors that are synaptic versus extrasynaptic. However, our data do not suggest that sNS
has caused any such shift in GluRs (see Fig 3).
Cornejo et al: Neonatal Seizures Alter Synapses
421
Fig 7. A single episode of neonatal seizures (sNS) increased the concentration of PSD-95 in adult CA1 hippocampus. Total hippocampal CA1 protein concentration (immunoreactivity/␮g protein) was generated by probing whole CA1 homogenates with antibody against PSD-95 (see Materials and Methods). (A) Typical anti–PSD-95 immunoblot shows marked increase in density after
sNS. (B) There was a significant increase in the total concentration of PSD-95 after sNS (1.084 ⫾ 0.127 immunoreactivity/␮g
protein; n ⫽ 8) compared with saline-injected control rats (0.757 ⫾ 0.076 immunoreactivity/␮g protein; n ⫽ 11; p ⫽ 0.008,
Student’s t test).
Fig 8. Schematic of proposed mechanisms causing altered plasticity after a single episode of neonatal seizures (sNS). Postsynaptic
changes in glutamate receptor (GluR) subunit numbers16 or properties17 are thought to underlie synaptic modification, which has
resulted in postulated “subunit rules”: (1) for GluR1-4, synaptic removal of GluR2 drags along GluR1 and GluR3 which causes
long-term depression (LTD); (2) GluR1 or GluR3 not associated with GluR2 (“homomers”) act independently; (3) insertion, modification, or both of GluR1 underlies LTP.20 Insertion of GluR1 predominates in long-term potentiation (LTP) at younger ages62
but is supplanted by modification at later ages.48 Preferential activation of different N-methyl-D-aspartate (NMDA) receptor subtypes is thought to underlie the induction of LTP (NR2A) versus LTD (NR2B).63– 65 GluR channels at excitatory CA1 hippocampal spiny synapses are formed by GluR1 (red bars) self-associating into homomers or in association with GluR2 (black bars) into
heteromers. After sNS, adult CA1 synapses have less of their total GluR1 expressed on the surface. As a result of this proportional
shift after sNS, GluR1s are more likely to be associated with GluR2s, but less total receptors are suggested to be present. Because of
equal excitability compared with naive synapses, it is postulated that potentiated GluR1s (asterisk) are stabilized by more relative
PSD-95 (filled spheres). By applying subunit rules (GluR1s mediate LTP and GluR2s drag along GluR1s for LTD) and noting
the loss of NR2A (activation favors LTP) and proportional gain of NR2B (activation favors LTD) after sNS, these synapses have
less LTP and more LTD.
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Alternatively, one might expect greater potential
movement of GluR1 to the membrane with LTP (see
Fig 8, “rule 3”); however, this is a feature of only immature synapses62 and not adult synapses.48 Even after
sNS the intracellular pool size is only 10% of the total
(see Fig 6A), thus permitting only limited LTP from
this pool, if it were involved. Nevertheless, any further
movement of GluR1s into the membrane may be prevented by the overexpression of PSD-95.59 Decreased
activation of NR2A because of its 28% loss also likely
contributes to the inability to induce LTP after sNS
based on pharmacological63,64 and biochemical data.65
Because of the intracellular shift of GluR1, remaining membrane-bound GluR1s are more likely to be associated with GluR2 after sNS. Because GluR2s drag
along GluR1s with LTD, greater LTD would be predicted in adult rats that experienced sNS (see Fig 8,
“rule 1”). Conversely, in naive synapses, some GluR1s
are likely to be unassociated with GluR2, and as a result (see Fig 8, “rule 2”), these independent GluRs will
not be removed, resulting in less LTD in naive synapses. PSD-95 overexpression favors greater LTD59 after sNS. It is also possible that there is a greater activation of NR2B both due to the 28% loss of NR2A
yet stable expression of NR1 and the increased expression of PSD-95 (which favors synaptic clustering of
NR2B50) after sNS. Greater activation of NR2B would
favor more LTD induction63,64 as a result of sNS.
Our data are consistent with enhanced LTD and absent LTP underlying impaired hippocampal-dependent
memory and supported by multiple lines of evidence.
Saturated LTP has been shown to impair hippocampaldependent learning.66 The RAWM has been shown to
be hippocampal dependent and capable of testing
memory.67 NMDA-R–mediated hippocampal LTP underlies spatial learning.41,68 However, when NMDA-R
were blocked and episodic-like memory was specifically
impaired, other plasticity mechanisms substituted (as
they likely did here) to allow MWM performance.29
Under certain conditions, NMDA-R–independent
forms of LTP induction are observed to coexist.69 In
future studies, it will be important to determine
whether these can be activated or enhanced to improve
learning and memory.
We did not find any evidence of synaptic reorganization or spine loss. It is possible that more subtle alterations in spine morphology occurred that could not
be detected with the techniques used. Severe cognitive
dysfunction, altered histology, and impaired plasticity
became apparent only after multiple episodes of earlylife seizure activity.5–7 Similarly, in other mouse models of developmental syndromes with learning impairment, altered spines were associated with either
abnormal postsynaptic responsiveness45 or more severe
learning impairment in the MWM,70 neither of which
we observed.
In a previous study of a single episode of early-life
seizures, increased inhibitory synaptic transmission was
demonstrated to mediate impaired LTP in the perforant path and DG.13 Similarly, in the Ts65Dn
mouse model of Down’s syndrome, increased inhibition has been shown to mediate impaired LTP.71 Notably, this was found primarily in the DG, whereas the
effect was much less pronounced in CA1,71 as studied
here. Altered inhibition in CA1 after a single episode
of early-life seizures could contribute and partially explain some of our findings. However, the pronounced
changes in glutamate receptors and PSD-95 (which is
not found in inhibitory synapses50) argue that the altered plasticity and memory that we observe are substantially mediated by alterations at glutamatergic synapses.
The effects of a single episode of early-life seizures in
rodent models are interrelated to the severity of the
seizures, developmental age, and technique. More severe and more prolonged seizures caused by bicuculline
at P4,72 direct intrahippocampal injection of kainate at
P7,73 intraperitoneal injection of lithium pilocarpine at
P1274 and up,75 prolonged electrical stimulation after
P14,76 or kainate at older ages51 are each associated
with overt cellular damage. Thus, our current demonstration that a single episode of relatively mild seizures
caused substantial, permanent functional alterations
without cellular damage now establishes with prior
studies13 the insidious nature of early-life seizures, but
uniquely implicates the damage to dysfunctional glutamatergic synapses.
It is important to distinguish whether we were inducing status epilepticus. Neonatal status epilepticus
has been found to be associated with an adverse clinical
outcome.3 Although this has been challenged,77 recent
studies suggest that outcome may be poor regardless of
classification.78 We do not think that the term status
epilepticus should be applied here for several reasons.
First, defining status epilepticus has been frequently
debated. In neonatal humans, the current definition is
for either: (1) electrical seizures lasting longer than 30
minutes, or (2) greater than 50% of the recording period occupied by ictal discharges.1,79,80 In rodents, discharges longer than 20 minutes have been taken as evidence of status epilepticus.11 In contrast, in adult
humans, discharges as brief as 5 minutes have been
considered to be a sign of status epilepticus.25 Second,
many resist applying the term status epilepticus to neonatal seizures, because in humans during this developmental time period, the level of consciousness cannot
be determined interictally.1,2 Even when human neonatal seizures are prolonged, as is often the case clinically, the term status epilepticus “should not be applied”
and a series of electrical seizures should be called a single clinical seizure.2 We suggest that our findings represent a rodent model of a single episode of relatively
Cornejo et al: Neonatal Seizures Alter Synapses
423
mild human neonatal seizures because the seizure duration in our hands does not experimentally fit a criterion of neonatal status epilepticus.
A better understanding of the developmental mechanisms of excitatory synaptic function that are both influenced and subsequently altered by sNS is necessary.
Recent studies suggest that our results at P7 could be
uniquely applicable to a developmental window equivalent to human neonatal seizures clinically,2 biochemically,23 and electrographically.24 As monitoring studies
have suggested that sNS induced by kainate does not
result in an epileptic state,9,11 we assume that our findings resulted from a single episodic insult. As we have
shown, novel approaches are required to begin to address effective treatment24 because conventional therapy24,81 is also inadequate clinically.82 Additional studies might be necessary to clarify the “dose–response”
relation between seizure severity and long-term outcome. Nevertheless, given the insidious problem of
neonatal seizure detection,78 our findings might suggest that neonates at-risk for seizures be prophylactically treated. However, defining risk and balancing
outcome with the potentially adverse consequences of
treatment83 requires further study. Through these additional characterizations, we expect that clinically relevant interventions to prevent the effects of sNS will be
forthcoming.
This work was supported by the NIH (National Institute of Mental
Health/APA Diversity Program in Neuroscience, MH18882-17,
B.J.C.; National Institute of Neurological Disorders and Stroke,
NS041267, T.A.B.).
We acknowledge E. Stubblefield, K. Manning, C. Algeier, Drs K.
Staley, M. Dell’Acqua, V. Dzhala, C. Adams, and J. Yonchek for
their contributions.
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