European Journal of Neuroscience, Vol. 23, pp. 3346–3358, 2006 doi:10.1111/j.1460-9568.2006.04865.

Environmental manipulations early in development alter

seizure activity, Ih and HCN1 protein expression later in life

Ulrich Schridde,1,2 Ulf Strauss,3 Anja U. Bräuer4 and Gilles van Luijtelaar2
1
Department of Neurology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520–8018, USA
2
NICI, Department of Biological Psychology, Radboud University Nijmegen, Nijmegen, The Netherlands
3
Neurobiological Laboratory, Clinic for Neurology, University of Rostock, Rostock, Germany
4
Department of Cellbiology and Neurobiology, Centrum of Anatomy, Humboldt University Hospital (Charité), Berlin, Germany

Keywords: absence epilepsy, hyperpolarization-activated cyclic nucleotide gated current, maternal deprivation, neonatal handling,
WAG ⁄ Rij rat

Abstract
Although absence epilepsy has a genetic origin, evidence from an animal model (Wistar Albino Glaxo ⁄ Rijswijk; WAG ⁄ Rij) suggests
that seizures are sensitive to environmental manipulations. Here, we show that manipulations of the early rearing environment
(neonatal handling, maternal deprivation) of WAG ⁄ Rij rats leads to a pronounced decrease in seizure activity later in life. Recent
observations link seizure activity in WAG ⁄ Rij rats to the hyperpolarization-activated cation current (Ih) in the somatosensory cortex,
the site of seizure generation. Therefore, we investigated whether the alterations in seizure activity between rats reared differently
might be correlated with changes in Ih and its channel subunits hyperpolarization-activated cation channel HCN1, 2 and 4. Whole-cell
recordings from layer 5 pyramidal neurons, in situ hybridization and Western blot of the somatosensory cortex revealed an increase
in Ih and HCN1 in neonatal handled and maternal deprived, compared to control rats. The increase was specific to HCN1 protein
expression and did not involve HCN2 ⁄ 4 protein expression, or mRNA expression of any of the subunits (HCN1, 2, 4). Our findings
provide the first evidence that relatively mild changes in the neonatal environment have a long-term impact of absence seizures, Ih
and HCN1, and suggest that an increase of Ih and HCN1 is associated with absence seizure reduction. Our findings shed new light on
the role of Ih and HCN in brain functioning and development and demonstrate that genetically determined absence seizures are quite
sensitive for early interventions.

Introduction
The Wistar Albino Glaxo ⁄ Rijswijk (WAG ⁄ Rij) rat is a valid genetic
model of absence epilepsy (Crunelli & Leresche, 2002; Coenen & van
Luijtelaar, 2003) in which seizures are characterized by spontaneous
generalized 7–9 Hz spike-wave discharges (SWD) in the EEG,
accompanied by decreased consciousness and the interruption of
ongoing activity.
Research in WAG ⁄ Rij rats has shown that absence seizures,
although of genetic origin, are sensitive to environmental manipula-
tions postweaning (Schridde & van Luijtelaar, 2004a). The nervous
system is especially susceptible to environmental influences early in
life (Andersen, 2003). It has been repeatedly shown that manipulations
such as neonatal handling (NH) or maternal deprivation (MD) lead to
permanently altered brain function and behaviour in adulthood (Hsu
et al., 2003; Pryce & Feldon, 2003). Therefore, we set out to
investigate environmental influences on absence seizures early in
development by manipulation of the neonatal rearing environment.
Various recent observations stress the role of the hyperpolarization-
activated cation current (Ih) in mediating seizure activity (Chen et al.,
2001; Brewster et al., 2002; Poolos et al., 2002; Bender et al., 2003;
Surges et al., 2003; Strauss et al., 2004; Budde et al., 2005; Poolos,
Correspondence: Dr Ulrich Schridde, 1Department of Neurology, as above.
E-mail: ulrich.schridde@yale.edu
U. Schridde, U. Strauss and A. U. Bräuer contributed equally to this work.
Received 29 June 2005, revised 10 March 2006, accepted 10 April 2006
2005). Ih is a mixed Na+ ⁄ K+ depolarizing current that is active at
membrane potentials important for integration of neuronal activity and
therefore contributes to various functions on the single-cell and on a
network level (Santoro & Tibbs, 1999; Chen et al., 2002). The
different properties of Ih are mediated by the particular assembly of
four channel constituting subunits (HCN1–4) and their specific
sensitivity to bind cyclic nucleotides like cAMP (Santoro et al.,
1997; Ludwig et al., 1998; Chen et al., 2002). Because HCN1–4
undergo changes during development (Santoro & Tibbs, 1999; Bender
et al., 2001; Vasilyev & Barish, 2002), and show plasticity after insults
(Chen et al., 2001; Shah et al., 2004) and lesions (Bräuer et al.,
2001a), we suggest that HCN channels and Ih might also be
susceptible to environmental influences. Indeed, it was recently shown
that neuronal Ih is sensitive for changes in housing conditions (Roberts
& Greene, 2005).
Especially in absence epilepsy, Ih seems to play a crucial role (Lüthi
& McCormick, 1998; Ludwig et al., 2003; Poolos, 2004, 2005; Budde
et al., 2005). In stargazer mice, absence seizures were associated with
alterations in Ih (Di Pasquale et al., 1997), and HCN2 knock-out mice
develop spontaneous absence seizures (Ludwig et al., 2003). In line
with this, we recently found an impaired Ih at the somatosensory
cortex of WAG ⁄ Rij rats (Strauss et al., 2004). In WAG ⁄ Rij rats the
somatosensory cortex is the site of seizure generation and spread
(Meeren et al., 2002), and pharmacological research suggests that this
might also hold for other absence seizure models such as Genetic
absence epileptic rats of Strasbourg (GAERS; Manning et al., 2004).

ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd

In pyramidal neurons from WAG ⁄ Rij rats the fast component of Ih
activation was strongly reduced resulting in a slower and smaller Ih,
compared to neurons from nonepileptic Wistar and August Copenha-
gen Irish (ACI) rats. This reduction in Ih was accompanied by a 21%
reduction of the HCN1 protein, while HCN1 mRNA was not altered
(Strauss et al., 2004).
The aim of the current study was to investigate the influence of the
neonatal rearing environment on absence seizures and Ih in WAG ⁄ Rij
rats later in life, using NH and MD. We first performed EEG
recordings in freely moving animals to establish putative influences of
environmental manipulations on SWD. Due to the close relationship
between SWD and behavioural states (Coenen et al., 1991; Drinken-
burg et al., 1991) and because NH or MD can influence behaviour
(Caldji et al., 2000), we also scored behaviour along with the EEG.
Next, we performed whole-cell recordings from layer 5 pyramidal
neurons of the somatosensory cortex, which represent the primarily Ih
and HCN1 expressing components of the neocortex (Lorincz et al.,
2002). This was combined with in situ hybridization and Western blot
to investigate whether differences in seizure activity might be
correlated with changes in Ih and HCN.
Materials and methods
Subjects and environmental manipulation
All experimental manipulations were in accordance with local
guidelines, approved by the ethical committee of Radboud University
Nijmegen and in agreement with the European Communities Council
Directive of 24 November 1986 (86 ⁄ 609 ⁄ EEC).
Prior to birth, pregnant WAG ⁄ Rij rats of our own colony were
randomly allocated to one of three neonatal rearing environments; no
treatment control, MD, or NH. All litters were born within a 9-day
period. No culling of the litters was applied to prevent handling effects
that result from the culling procedure itself (Weizman et al., 1999).
Day of birth was designated postnatal day (PN) 0. NH and MD were
carried out daily between the fourth and seventh hour of the light
phase, starting on PN1 and continued till PN21 (day 22). Rats of the
control group were left undisturbed during this time, except one day
for cage maintenance. For NH and MD the mother was first removed
from its litter and temporary separated in a clean cage (always the
same cage for each mother). In case of NH each pup was transferred to
a small plastic box filled with sawdust and lined with paper towels,
where it remained for 15 min before being transferred back to the
home cage, followed by the mother. In case of MD, the pups remained
in their home cage for 180 min before reunion with their mother.
Around 30 days of age the rats were weaned and separated by sex.
Only male rats were randomly selected. From then on rats were
housed in pairs (same treatment, different litter) in standard macrolon
Type III cages on a reversed 12-h light : 12-h dark cycle (light on at
21:00 h) with food and water ad libitum in a temperature controlled
room. Rats were left undisturbed except for routine cage maintaining
purposes. All measurements were performed in adult rats of 4½
months and older, when SWD are fully developed and frequently
present in the EEG of all WAG ⁄ Rij rats (Coenen & van Luijtelaar,
2003). The mean age and SEM of the rats in days per group were
190.55 ± 19.65 for control, 201.73 ± 20.78 for MD, and
191.70 ± 18.71 for NH, respectively (P ¼ 0.91).
Surgery
Rats were anaesthetized with isoflurane and mounted in a stereotaxic
frame (Kopf Instruments, Tujunga, CA, USA). Atropine (0.1 mL,
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 23, 3346–3358
Environment alters absence seizures, Ih, and HCN1 3347
i.m.) was applied as a parasympatholytic agent to prevent excessive
salivation and 2% lidocaine for local analgesia. Maintenance anaes-
thesia was provided via permanent isoflurane inhalation applied
through a nose mask and periodically assessed by limb withdrawal
reflex. Body temperature was monitored via a sensing probe and
maintained at 37
C using a heating pad. Rats were equipped with
unilateral epidural electrodes (Plastic One MS-333 ⁄ 2-A, Roanoke,
VA, USA) over the frontal (A-P 2.0; M-L 3.5) and parietal (A-P )6.0;
M-L 4.0) cortex. An electrode placed over the cerebellum served as
ground. All coordinates were determined according to Paxinos &
Watson (1998) (Bregma zero-zero, skull surface flat). The electrode
socket was fixed to the skull using two stainless steel jeweler screws
and dental acrylic. The tissue was sutured and rats were allowed to
recover for approximately 14 days.
Electroencephalographic and behavioural recordings
For EEG recordings 16 rats of each group (control, MD, NH) were used.
First, rats were transferred to Plexiglas recording cages
(25 · 30 · 35 cm), equipped with food and tap water ad libitum, and
connected to EEG leads via a swivel that allowed free movement. The
signals were amplified and filtered (1–100 Hz), digitized at a sampling
rate of 512 Hz and saved on disk for off-line analysis using a
microcomputer and a Windaq system (DATAQ Instruments, Akron,
OH, USA). The rats were given one day to habituate to the recording
situation to prevent any putative confounding novelty effects on the
EEG recordings. The EEG was recorded for four hours during the dark
phase between 10:00 h and 14:00 h, when the number of paroxysms is
quite high (van Luijtelaar & Coenen, 1988). Simultaneously with the
EEG, the animals’ behaviour was recorded on video for off-line analysis.
During recordings rats were exposed to background white noise
(53.2 dB).
EEG and behavioural analyses
SWD were detected automatically using SWC-finder (written by PLC
van den Broek) and subsequently visually confirmed by one of the
authors. This was carried out to prevent the occurrence of false positives
(e.g. sleep spindles, artifacts) or false negatives (SWD that were not
identified by SWC-finder, but could clearly be identified as SWD upon
visual inspection by a qualified scorer). Although WAG ⁄ Rij rats show
two types of SWD (van Luijtelaar & Coenen, 1986; Schridde & van
Luijtelaar, 2004b) in the current study only the absence seizure related
Type 1 SWD (here simply referred to as SWD) were scored and
analysed. The following criteria were used for SWD identification (see
also Fig. 1B); a clear asymmetric 7–9 Hz spike and wave pattern with a
spike amplitude of at least twice the background activity and a duration
of 1 s or longer (van Luijtelaar & Coenen, 1986). The total number and
mean duration of SWD were determined for the four hour recording
period. According to criteria defined elsewhere (van Luijtelaar et al.,
1996), exploratory (sniffing, rearing, walking, digging, and manipula-
ting objects with the forepaws), automatic (grooming, eating, drinking,
licking, and defecation) and passive (sitting, standing or lying still, and
sleeping) behaviours were scored from video during the first 15 min of
each consecutive hour, using a Tandy 102 and The Observer (Noldus,
Wageningen, The Netherlands) for post processing (Noldus, 1991). The
total duration (in seconds) of each behaviour was determined per 15-min
interval. All scorings (EEG and behaviour) were conducted blind with
respect to treatment condition.
All statistical analyses were run in SPSS (v. 11.5, SPSS Inc.,
Chicago, Il, USA) using one-way anova or manova for repeated
3348 U. Schridde et al.

Fig. 1. Early environmental manipulations influence the number of spike-wave discharges in adult rats. (A) Examples of EEG recordings from adult rats of the
three different neonatal rearing environments, illustrating that rats that underwent maternal deprivation (WAG ⁄ Rij MD) or neonatal handling (WAG ⁄ Rij NH) show
less spike-wave discharges (SWD) in their EEG compared to normal reared controls (WAG ⁄ Rij control). The bar in WAG ⁄ Rij control indicates section of expanded
SWD shown in B. (B) A typical SWD as recorded in WAG ⁄ Rij rats. A SWD is characterized by a pattern of asymmetric sharp spikes with an amplitude of at least
twice the background EEG activity, followed by a slow wave. SWD have a frequency around 7–9 Hz. In A and B positivity is upward. (C) Shows difference
between neonatal rearing environment groups for number of SWD. Rats that underwent maternal deprivation (WAG ⁄ Rij MD) or neonatal handling (WAG ⁄ Rij NH)
during the first three weeks of life showed as adults less SWD during the four-hour recording period, compared to normal reared control rats (WAG ⁄ Rij control).
*P < 0.011, indicates level of significance for difference between neonatal rearing environment groups, as revealed by one-way anova. (D) The mean duration (in
seconds) of SWD was not different between groups. In C and D bars represent mean ± SEM.

measures followed by Duncan posthoc test if appropriate. The
between factor for all analyses was neonatal rearing environment
(control, MD, and NH). A P < 0.05 was regarded as significant.
Slice preparation
Acute coronal brain slices were prepared from adult WAG ⁄ Rij rats
(ncontrol ¼ 6, nMD ¼ 7, nNH ¼ 7) as described (Strauss et al., 2004).
Rats were deeply anaesthetized with ether and decapitated. The brain
was removed and immediately transferred to cold (2–5
C), carbo-
genated (95% O2, 5% CO2), artificial cerebrospinal fluid (ACSF)
containing (in mm): 124 NaCl, 3.5 KCl, 1.25 NaH2PO4, 2 MgSO4,
26 NaHCO3, 10 D (+)-glucose and 2 CaCl2 (all chemicals were of
analytical grade, Merck, Darmstadt, Germany). Using a vibrating
microtome (TPI 1000, Vibratome, St. Louis, MO, USA), brain slices
of 400-lm nominal thickness were cut and allowed to recover for at
least 1 h at 33 ± 1
C before they were stored at room temperature.
Patch-clamp recordings
Individual slices were transferred to a submerged recording chamber
(1–2 mL ⁄ min ACSF at 34
C) and pyramid-shaped layer 5 neurons of
the somatosensory cortex were visualized using infrared differential
interference contrast (IR-DIC) video microscopy with a ·40 magni-
fication water immersion objective (Axioskop 2 FS Zeiss, Göttingen,
Germany). Somatic whole-cell current and voltage recordings were
performed as described for layer 2 ⁄ 3 neurons (Strauss et al., 2004).
Patch pipettes were pulled from corning #0010 glass capillaries (WPI,
Berlin, Germany) to a final pipette resistance between 3 and 5 MW.
During voltage clamp, maximum tolerated series resistance was
15 MW, with changes < 20% during recordings. There was no
significant difference in series resistance between recordings of
control, MD or NH neurons. The pipette solution contained (in
mm): 120 K-methylsulphate, 20 KCl, 14 Na-phospocreatine,
0.5 EGTA, 10 HEPES, 4 Mg2+-ATP, and 0.3 Tris2GTP (Sigma-Ald-
rich, Steinheim, Germany) (pH 7.25, 288 mOsm). An empirically
calculated liquid junction potential of )10 mV has not been corrected
for.
Voltage-clamp recording of pharmacologically isolated Ih was
performed after blocking the fast inward-rectifying potassium chan-
nels (KIR) with 400 lm Ba2+ (Merck) added to a modified ACSF
(referred to as divalent cation solution – DCS) with MgSO4 replaced
by MgCl2, and with NaH2PO4 omitted. Na+-currents were blocked
with 0.5 lm tetrodotoxin (TTX, Tocris, Bristol, UK) and Ca2+-
currents with 1 mm Ni2+. In some experiments 20 lm 6-cyano-7-

ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 23, 3346–3358

nitroquinoxaline-2,3-dione (CNQX), 20 lm d(–)-2-amino-5-phospho-
nopentatonic acid (D-APV) and 10 lm bicuculline were added to
block spontaneous synaptic events. To block Ih, the specific antagonist
4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chlor-
ide (ZD7288, Tocris) was prepared as a 100 mm solution, divided in
aliquots and kept at )20
C until use, when it was dissolved to its final
concentration (25–50 lm) in the modified ACSF.
Data from patch-clamp recordings were collected with an EPC-10
(HEKA, Lambrecht, Germany), digitized (50 kHz, after Bessel
filtering at 5.0 kHz) and stored using PULSE software (HEKA). In
current-clamp whole-cell mode the following characteristics were
gathered. The membrane resistance (RN) was estimated by linear
regression of the steady state I–V curve in the close proximity
(± 5 mV) of the resting potential (VM). The membrane time constant
(sM) was determined as the slowest component of a multiexponential
least square fit of the initial 200 ms of the voltage response to current
steps near (± 5 mV) VM. The relationship between injected current
intensity and the total number of spikes per pulse (normalized by the
pulse duration) was calculated, and neurons without a significant
correlation between these variables were excluded from analysis. The
current–frequency relationship of a given neuron is characterized by
the slope (Hz ⁄ nA) of the linear regression.
After cancelling the fast (pipette) capacitance in the voltage-clamp
cell-attached mode, the cell capacitance (CM) was obtained in the
whole-cell mode from the integral of the slow capacitive transient
from 10 mV voltage steps with alternating de- and hyperpolarizing
polarity from a holding potential of )50 mV to minimize the influence
of Ih on the measurement. The tail current amplitudes (a1) and
maximal tail current (a2) from a )130 mV step were plotted against
the preceding step voltage (V) and well fitted separately for each
neuron with the Boltzmann equation of the form:
IðV Þ ¼ ða1
a2 Þ=½1 þ a2 þ eðV
V 1=2 Þ=k  ð1Þ
V1 ⁄ 2, the midpoint of voltage activation, and k, the slope-factor of the
curve, were obtained to identify the voltage sensitivity of the
h-channel. Double-exponential fits (excluding an initial delay of 10–
30 ms) of the trajectory of the Ih traces yielded first-order (sfast) and
second-order (sslow) time components. The amplitude of Ih was
obtained with a ZD7288 subtraction method and ⁄ or leak subtraction
protocols and ⁄ or back-extrapolating double-exponential fits to the
start of the current with highly similar results (Sciancalepore &
Constanti, 1998; Bräuer et al., 2001a; Strauss et al., 2004). The
reversal potential was determined by stepping to different test
potentials after fully activating the Ih (step potential )140 mV for
2 s). Linear regression of the tail currents (measured as described for
voltage sensitivity) yielded the reversal potential.
Analysis and exponential fits of the current and voltage traces were
performed with PULSE-FIT 8.63 (HEKA). Paired and unpaired
Student’s t-tests or one-way-anova were performed with the SPSS
software package (v. 10.0, SPSS Inc., Chicago, Il, USA). All data are
presented as mean ± SEM.
In situ hybridization
Six brains per group from adult WAG ⁄ Rij (ncontrol ¼ 6, nMD ¼ 6,
nNH ¼ 6) were dissected and frozen on dry ice. Horizontal slices
(19 lm) were cut on a cryotome and fixed in 4% paraformaldehyde
(w ⁄ v), washed in 0.1 m PB (pH 7.4), and dehydrated afterwards. An
antisense oligonucleotide (59-CCGATCGAGTCGGTCAATAGC-
AACTGTCTCAAAGGCTCTTCTCATCAT-39) complementary to
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 23, 3346–3358
Environment alters absence seizures, Ih, and HCN1 3349
bases 1923–70 of HCN1 cDNA (GenBank acc. # AJ225123) was
used. The specificity of the oligonucleotide was confirmed by a
BLAST GenBank search and showed no significant cross matches
with any known sequences and ESTs. The in situ hybridization was
performed as described in Bräuer et al. (2001b). The oligonucleotides
were end labelled using terminal deoxynucleotide transferase (Boeh-
ringer Mannheim, Mannheim, Germany) and [a-35S]dATP (DuPont
NEN, Boston, MA, USA); 400 000 cpm labelled oligonucleotide in
50 lL hybridization buffer was used per section. Hybridization was
performed for 16 h at 42
C in a humidified chamber, then the slides
were washed for 2 · 30 min in 0.1 · SSC at 56
C and 1 · 10 min in
0.05 · SSC at room temperature (RT). Finally, the sections were
rinsed in H2O at RT and dehydrated. For autoradiography, slides were
exposed to Kodak X-OMAT AR X-ray films for 14 days. No signals
were detected on sections hybridized with a specific antisense
oligonucleotide when unlabelled oligonucleotide was added in 100-
fold surplus. A detailed description of the antisense oligonucleotides
and the protocol used can be found elsewhere (Bräuer et al., 2001a, b).
Western blot
For Western blot analysis total protein extracts of the neocortex from
brains of adult WAG ⁄ Rij rats (ncontrol ¼ 3, nMD ¼ 3, nNH ¼ 3) were
used. The brain of each WAG ⁄ Rij rat was rapidly dissected isolating
the neocortex. Brain regions were disrupted using a glass-Teflon
homogenizer in five-fold (volume ⁄ weight) lysis buffer (20 mm Tris,
pH 7.5 0.25 m sucrose, 1 mm EGTA, 5 mm EDTA, 1 mm PMSF,
50 mm b-mercaptoethanol, 25 lg ⁄ mL leupeptin, 10 U DNase I,
10 U Rnase A, 250 lg ⁄ mL lysozyme]. Homogenates were spun for
20 min at 1000 · g, the supernatants were saved. The protein
concentrations of the supernatants were determined using the
bicinchoninic acid (BCA) technique (Pierce, Rockford, Il, USA),
and 40 lg was loaded per lane from each neocortex in duplex and
were separated using sodium dodecyl sulphate–polyacrylamide gel
electrophoresis (SDS–PAGE) and transferred to nitrocellulose (Sch-
leicher & Schuell, Dassel, Germany). Immunoanalysis was performed
with anti-HCN-1 (diluted 1 : 200), anti-HCN-2 (diluted 1 : 200), and
anti-HCN-4 (diluted 1 : 200) polyclonal antibodies all from Alomone
Laboratories (Jerusalem, Israel). After blocking, each blot was probed
with appropriate primary antibodies in blocking buffer, followed by
sheep anti-rabbit IgG conjugated to horseradish peroxidase (1 : 5000)
(Amersham, Buckinghamshire, UK). The final protein IgG complexes
were visualized following the reaction to 3,3¢-diaminobenzidine
tetrahydrochloride (Amersham). As an internal control for standard-
ization of protein amount b-actin (Sigma) expression was used
throughout. The prestained protein Marker (New England Biolabs,
Ipswitch, MA, USA) was used as protein molecular size reference.
Quantification of Western blot analysis
Quantifications of Western blots were performed using a computerized
videodensitometry system (Metamorph, Universal Imaging, Downing-
town, PA, USA). All measurements were focused at the neocortex. The
autoradiographs were digitalized using a scanner (Micotek ScanMak-
er 636). For analysis of the intensity of each band, the computerized
video densitometry system was used to quantify the signal intensity on a
pixel level. A visually established pixel intensity threshold was set to
remove the unlabelled portion of the image. A standard rectangle
(1.5 mm2) was defined and placed at three different positions over the
band. For each position, the percentage of pixels within the rectangle
representing signal intensities higher than the threshold was determined
3350 U. Schridde et al.

without further discriminating between signal intensities above this

value. For normalization the HCN band was normalized to the
correlative b-actin band in each lane. Statistical analyses were run in
SPSS (v. 11.5, SPSS Inc., Chicago, Il, USA) using one-way anova,
followed by Scheffe posthoc test if appropriate. The between factor for
all analyses was neonatal rearing environment (control, MD, and NH). A
P < 0.05 was regarded as significant.

Results
Early environmental manipulations influence SWD in adult rats
Independent of neonatal rearing environment, all WAG ⁄ Rij rats
showed SWD (Fig. 1A and B). A SWD was characterized by a pattern
of sharp positive spikes followed by a slow wave. The amplitude of
the spikes was at least twice the normal background EEG activity and
SWD had a frequency around 7–9 Hz. Although all rats developed
seizures, we observed a marked difference in the number of seizures
between groups during the four-hour recording period (F2,38 ¼ 5.06,
P ¼ 0.011). The Duncan posthoc test revealed that WAG ⁄ Rij rats that
underwent NH or MD had significantly less SWD (21 ± 3.48 SEM,
NH; 25.08 ± 2.91 SEM, MD) compared to rats of the control group
(35.71 ± 3.71 SEM). Compared to rats from the control group, NH
and MD rats had on average 35% less discharges, while the latter two
did not differ (Fig. 1C). In contrast to the number of SWD, we found
no effect of early environmental manipulations on the mean duration
of SWD (F2,38 ¼ 1.14, P ¼ 0.33); WAG ⁄ Rij control (2.78 s ± 0.11
SEM), WAG ⁄ Rij MD (2.63 s ± 0.11 SEM), and WAG ⁄ Rij NH
(2.54 s ± 0.13 SEM) (Fig. 1D).
To control for the possibility that the observed decrease in seizure Fig. 2. Early environmental manipulations have no effect on behaviour.
activity in NH and MD rats was confounded by a putative increase in (A) Time spend by control (WAG ⁄ Rij control), maternal deprivation
activity, we also scored behaviour during EEG recordings. The (WAG ⁄ Rij MD), or neonatal handling (WAG ⁄ Rij NH) rats in explorative,
automatic or passive behaviour during EEG recordings, respectively. Although
repeated-measurements manova revealed effects for behaviour
the time spent by the rats in the various behavioural categories differs, there
(F2,37 ¼ 393.06, P < 0.001), indicating that the various behavioural were no differences between treatment groups (WAG ⁄ Rij control, WAG ⁄ Rij
categories were differently present during the recording period, and an MD, WAG ⁄ Rij NH). This demonstrates that early environmental manipulations
interval by behaviour interaction effect (F6,33 ¼ 6.88, P < 0.001) did not affect behaviour. (B) Behaviour scores over the four 15-min intervals
(Fig. 2A and B). However, we did not find any differences in of each consecutive hour to illustrate the interval by behaviour interaction
effect. Because no effects were found for early environmental manipulations,
behaviour between control, NH or MD WAG ⁄ Rij rats, showing that behavioural data were pooled. *Indicates difference compared to automatic
early environmental manipulations did not affect behaviour in the behaviour; **indicates difference compared to passive behaviour;  indicates
recording cage during EEG recordings (Fig. 2A). Further analysis of difference compared to automatic and passive behaviour, as revealed by
the interval by behaviour interaction effect showed that passivity, repeated measurements manova. In all cases P < 0.001. All data represent
total duration in seconds (mean ± SEM) either over the whole recording period
although generally the dominating behaviour, declined over time,
(A) or per interval (B).
while automatic and explorative behaviours increased. Significant
differences between behavioural categories were found across inter-
vals, with passive > automatic > explorative behaviour during the first WAG ⁄ Rij rats under the same conditions (Table 1). Consequently the
(F2,39 ¼ 952.84, P < 0.001) and second (F2,39 ¼ 61.38, P < 0.001) neurons of WAG ⁄ Rij control rats possessed an increased excitability
interval and passive, automatic > explorative behaviour during the in response to current injections due to an increase in neuronal gain
third (F2,39 ¼ 238.93, P < 0.001) and fourth (F2,39 ¼ 40.96, and due to an offset in the input output relationship (Fig. 3, Table 1).
P < 0.001) interval (Fig. 2B). Differences in neuronal resting potentials (VM) in the three groups
only became apparent after blocking fast inward rectifying and Ca2+-
activated K+, as well as TTX sensitive Na+-currents (Table 1). The
Increased Ih in layer 5 neocortical pyramidal neurons in adult differential influence of Ih on these VM group differences could be
NH and MD rats confirmed by a more pronounced effect of ZD7288 in the maternal
A comparison of voltage behaviours of pyramidal neurons in layer 5 deprived and the neonatal handled group (Table 1). The differences in
from the somatosensory cortex of WAG ⁄ Rij rats reared in the three the voltage responses to hyperpolarizing current steps in whole cell
neonatal environments is shown in Fig. 3 and Table 1. Neurons from current clamp recordings pertain to the classical hallmarks of Ih,
nontreated control WAG ⁄ Rij rats had a higher input resistance (RN) namely the voltage sag during hyperpolarizing current injections and
compared to those from rats which were neonatally handled or the subsequent transient rebound depolarization (Table 1). In neurons
maternal deprived, when superfused with ACSF and DCS, but not from MD and NH rats the voltage sag was pronounced and faster in
when superfused with ZD7288, a selective Ih blocker (Harris & onset than in neurons from control animals, which also had a less
Constanti, 1995) (Table 1). This was accompanied by a prolonged prominent rebound depolarization. In line with this, application of
membrane time constant (sM) of neurons from nontreated control 50 lm ZD7288 increased the input resistance to an equal level in all

ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 23, 3346–3358
 Environment alters absence seizures, Ih, and HCN1 3351

We further explicitly and directly determined the functional

Fig. 3. Comparison of voltage responses of representative layer 5 pyramidal
neurons at the somatosensory cortex of rats from different neonatal rearing
environments. The whole-cell current-clamp traces show voltage responses to
500-ms long current steps (depicted below the respective voltage traces, second
and fourth row, frequency 0.1 Hz). Top, voltage responses to a 0.2 nA
suprathreshold current injection. Middle, traces recorded in the presence of
400 lm Ba2+ to block fast inward rectification, 1 mm Ni2+ to block calcium
currents and 1 lm TTX to block sodium currents provide an insight into
differences in voltage sag during hyperpolarization and posthyperpolarization
rebound depolarization. Bottom, traces recorded in the presence of the specific
Ih blocker ZD7288 (50 lm, 15 min). Note that in NH this amount of ZD was
not sufficient to fully block the voltage sag. The scale bars apply to all three
groups. The dotted line represents a membrane potential of )65 mV. Note the
pronounced hyperpolarization following ZD7288 application in MD and NH
neurons.
three groups (Table 1), diminished the voltage sag, prevented the
rebound depolarization, and hyperpolarized the resting potential in
neurons of rats from all three rearing environments (control, MD,
NH), but the effect was pronounced in neurons of MD or NH rats. All
these observations suggest a greater contribution of Ih to membrane
voltage responses in rats that underwent MD and NH.
expression of Ih by somatic whole-cell voltage-clamp recordings
under conditions to minimize the contribution of other voltage gated
currents (see Materials and methods). Representative recordings of Ih
in layer 5 somatosensory pyramidal neurons are presented in Fig. 4A.
As the voltage sag the remaining inward current could be largely,
slowly and irreversibly inhibited by extracellularly applied ZD7288
(50 lm), confirming its identity as Ih (Fig. 4A). Ih increased in
amplitude after both types of early postnatal manipulation, but more
pronounced in NH (I ¼ )517.72 ± 81.57pA, n ¼ 10) than in MD
(I ¼ )396.68 ± 45.47 pA, n ¼ 12), compared to I ¼ )302.21 ±
68.95 pA (n ¼ 9, P < 0.05, respectively) in normal reared control
WAG ⁄ Rij rats (measured as steady state current close to the end of a
2-s voltage step to )130 mV). Whole cell current–voltage relation-
ships are depicted for the population in Fig. 4B. The increase in
amplitude appeared more pronounced over a broad voltage range
when taking approximations of the individual cell sizes into account,
i.e. when comparing the current densities, computed as Ih ⁄ Cm (current
amplitude normalized to cell capacitance, Fig. 4C; P < 0.05 between
)80 and )130 mV for MD and NH compared to control,
respectively). For voltage steps to )130 mV the current densities are
approximately doubled in MD (Ih ⁄ Cm ¼ )4.19 ± 0.43 pA ⁄ pF,
n ¼ 12) and NH (Ih ⁄ Cm ¼ )4.72 ± 0.59 pA ⁄ pF, n ¼ 10) compared
to normally reared control WAG ⁄ Rij rats (Ih ⁄ Cm ¼ )2.31 ±
0.25 pA ⁄ pF, n ¼ 9). A closer look at the physiological range in
which Ih will shape the excitability of neurons, i.e. at a membrane
potential of )80 mV, revealed that current density also doubled
following either MD or NH. The current density data suggest larger
h-channel availability or a higher single h-channel conductance in the
neurons of the rats which underwent MD or NH.
Additionally, Ih activation was sensitive to the rearing conditions;
i.e. became more rapid following MD or NH as is evident in Fig. 5A
and B. To characterize Ih activation in more detail, the Ih waveform
was fit with the sum of two exponential functions, which were
necessary and sufficient to adequately describe the activation kinetics
as shown superimposed in Fig. 5A. The fast (sfast) and slow (sslow)
time constant differed by approximately one order of magnitude and
were voltage dependent for all rearing environments (data not shown).
The sfast displayed acceleration with the change in rearing environ-
ments. At )130 mV sfast was consistently slower in normally reared

Table 1. General current-clamp properties of layer 5 pyramidal neurons from somatosensory cortex of WAG ⁄ Rij rats raised under different rearing conditions

Control (n) MD (n) NH (n)

RMP (mV)
ACSF )68.1 ± 0.78 23 )68.4 ± 0.78 23 )69.5 ± 0.88 21
DCS )64.2 ± 0.92 12 )60.4 ± 1.15* 15 )60.6 ± 1.1* 12
ZD7288-DCS 0.8 ± 0.81 6 )4.2 ± 1.5* 9 )5.1 ± 1.17* 6
RN (MW)
ACSF 201.7 ± 14.3 23 150.0 ± 8.8* 23 119.0 ± 15.2** 21
DCS 264.4 ± 15.9 12 212.1 ± 15.6* 15 177.0 ± 17.3** 12
ZD7288 311.9 ± 39.0 6 331.6 ± 48.0 9 307.6 ± 82.1 6
sM (ms)
ACSF 29.2 ± 1.69 23 22.4 ± 1.27** 23 21.6 ± 1.22** 21
DCS 47.2 ± 1.7 12 38.9 ± 2.71* 15 36.9 ± 1.5* 12
ZD7288 55.6 ± 5.1 6 60.2 ± 4.54 9 58.3 ± 4.91 6
‘Sag’ at )100 ± 3 mV (mV) )4.1 ± 0.88 12 )6.9 ± 0.7* 15 )7.8 ± 0.77** 12
Afterpotential post )100 ± 3 mV (mV) 3.6 ± 0.44 12 5.6 ± 0.48** 15 7.2 ± 0.34** 12
I–ƒ slope (Hz ⁄ nA) 90.9 ± 7.76 23 68.1 ± 4.81* 23 71.1 ± 4.01* 21
Rheobase – action potential current threshold (pA) 38.2 ± 5.24 23 76.4 ± 9.49* 23 86.7 ± 15.83* 21

Data presented as the mean ± SEM (n, number of cells), *P < 0.05 and **P < 0.01 when compared to neurons from nontreated WAG ⁄ Rij rats (control). Note that
I–ƒ slopes and rheobases were gathered in ACSF, whereas ‘sags’ and afterpotentials are taken from measurements in DCS. ZD7288-DCS indicates the difference
between the respective membrane potentials.

ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 23, 3346–3358
3352 U. Schridde et al.
WAG ⁄ Rij rats (131.6 ± 17.7 ms, n ¼ 9) than following NH
(84.2 ± 11.6 ms, n ¼ 10) or MD (80.5 ± 10.6 ms, n ¼ 12)
(Fig. 5C). No changes were detected for sslow (900 ms for the
control WAG ⁄ Rij, 1020 ms for the NH, 860 ms for the MD, one
way anova, P > 0.2, respectively). One should be aware that in
somatic recordings currents properly reflect channel activity only in
somatic and proximal dendritic membrane and therefore kinetic values
are solely used for comparative reasons and can not be taken as
absolute values.
Both changes, the increase in Ih amplitude and the acceleration of
its activation could in principle be caused by a shift in the voltage
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
dependence of Ih towards positive potentials. Therefore we analysed
steady state voltage activation of Ih in layer 5 neurons of rats from
all three rearing environments separately for each neuron (see
Materials and methods). Returning to a VM of )50 mV after voltage
steps of 2 s ()130 to )40 mV) revealed a gradual decline in the
tail current. The deactivation time constant (Fig. 5D and F) revealed
by monoexponential fits was slower in neurons from normally
reared control WAG ⁄ Rij rats (sdeactivation ¼ 246 ± 53.9 ms, n ¼ 9)
than following NH (sdeactivation ¼ 141.6 ± 42.9 ms, n ¼ 10,
P < 0.05) or MD (sdeactivation ¼ 145.4 ± 38.1 ms, n ¼ 12,
P < 0.05). As deactivation of Ih appeared relatively slow in all
three groups, the relative conductance was determined from tail
current amplitudes measured 25 ms after repolarization (vertical
dotted lines in Fig. 5D, for details see Materials and methods).
Normalized tail currents could be well fitted with a Boltzmann
equation which yielded similar V1 ⁄ 2 and k between groups (Fig. 5E,
Table 2).
Putative changes in basal modulation of Ih by different resting levels
of cyclic nucleotides in the three groups were explored using the
intracellular presence of a saturating concentration of cAMP (100 lm)
in an additional set of experiments. The application of cAMP slightly
shifted the V1 ⁄ 2 by only 1–3 mV, but markedly increased the slope-
factor of the voltage sensitivity in all three groups (Fig. 5E, Table 2).
However, intracellular cAMP did not lead to apparent group
differences in the slope-factor of the voltage sensitivity. There was
no change in the rate of activation or deactivation of Ih (Fig. 5C and
F). This is consistent with the predominant expression of HCN1 in
neocortical pyramidal neurons described by many investigators
(reviewed in Robinson & Siegelbaum, 2003). However, the group
differences in Ih amplitude and density between normally reared
WAG ⁄ Rij rats (control) on one hand and MD and NH on the other
hand were maintained (Fig. 4B and C). For voltage steps to )130 mV
the current densities are again approximately doubled in MD
(Ih ⁄ Cm ¼ )3.8 ± 0.33 pA ⁄ pF, n ¼ 10) and NH (Ih ⁄ Cm ¼ )4.3 ±
0.45 pA ⁄ pF, n ¼ 9) compared to normally reared control WAG ⁄ Rij
rats (Ih ⁄ Cm ¼ )2.46 ± 0.24 pA ⁄ pF, n ¼ 8). Therefore, it is unlikely
that any of these group differences are due to kinase independent
actions of cAMP on HCN channels (Wainger et al., 2001). The
Fig. 4. Increase of Ih in adult rats after early environmental manipulations.
(A, left) Families of Ih traces recorded in somatic whole-cell voltage-clamp
from the layer 5 pyramidal neurons of the somatosensory cortex shown current
clamped in Fig. 3. Neonatal rats underwent maternal deprivation (WAG ⁄ Rij
MD), neonatal handling (WAG ⁄ Rij NH) or were reared normally (WAG ⁄ Rij
control). Currents were elicited by 2-s-long voltage steps from )40 mV to
30 mV from a holding potential of )50 mV as depicted at the bottom. All
recordings were made in the presence of 400 lm Ba2+ (to block IKir), 1 mm
Ni2+ (to block ICa) 1 lm TTX, CNQX, D-AP5, and bicuculline. When
comparing data achieved in current-clamp (Fig. 3) and in voltage-clamp it is
important to note that the amount of Ih critically determines the input resistance
of, and electrotonic distance in, a given neuron and therefore the size of the sag
alone is not representative for the amount of Ih. Right, inhibition of Ih by
ZD7288. Displayed are current traces elicited by hyperpolarizing pulses of
)140 mV, as depicted at the bottom, before and 15 min after superfusion with
the above mentioned bath solution containing 50 lm ZD7288. Both, steady
state and tail currents were largely inhibited in all three groups. (B) Popula-
tion data illustrating an increase in Ih amplitude in NH and MD rats. The steady
state amplitudes (at times indicated by the dotted lines in A) were used for
construction of average I–V relationships. (C) Steady state amplitudes of every
single neuron were normalized to the individual cell capacitance, subsequently
averaged and given as current densities. Note that Ih densities do not differ in
neurons of rats from the same rearing group, whereas the differences between
control and MD or NH became more pronounced for this parameter. For B and
C control (n ¼ 9), control cAMP (n ¼ 8), MD (n ¼ 12), MD cAMP (n ¼ 10),
NH (n ¼ 10), NH cAMP (n ¼ 9). Data are displayed as means ± SEM.
European Journal of Neuroscience, 23, 3346–3358

Fig. 5. Acceleration of Ih activation after maternal deprivation and neonatal
handling, but no change in voltage dependence of Ih. (A) Traces (grey) represent
the current response from neurons of nontreated WAG ⁄ Rij rats (control), and rats
that underwent maternal deprivation (MD) or neonatal handling (NH) to a
hyperpolarizing voltage pulse of )120 mV (bottom). The traces are capacity and
linear current subtracted as described in Material and methods. Ih waveforms are
well fitted by the sum of two exponential functions as shown superimposed
(black) on the traces. (B) The first part of the traces depicted in A (dotted lines
demonstrate the section in the voltage protocol at the bottom) was normalized to
steady state amplitude to illustrate the acceleration of the current activation.
(C) Quantitative comparison of the fast activation time constants (sfast activation)
in response to a )130 mV voltage step without (solid diamonds) and with (open
diamonds) 100 lm cAMP in the pipette solution. Note the slowing of the initial
fast component of Ih in the control when posthoc compared to MD or NH.
(D) Representative families of tail currents recorded with cAMP free pipette
solution upon return to )50 mV from 2-s voltage steps from )130 to )40 mV (for
the voltage protocol see bottom left, section indicated by dotted lines). Note the
reduced tail amplitudes and the slower deactivation of Ih in neurons of control rats
(left). Tail current amplitude measurements were taken at the time indicated by the
vertical dotted lines marked with circles. (E) Population data of voltage
dependence depicted as mean relative tail currents, plotted against the preceding
test potential without (left panel, circles) and with (right panel, triangles) 100 lm
cAMP in the pipette solution. Curves were fitted with a Boltzmann function (lines
and dotted lines, see Materials and methods). For the fitting parameter,
particularly V1 ⁄ 2 and k, see Table 2. (F) Population data for deactivation time
constants (sdeactivation) estimated at a step back to )50 mV from a 2-s )130 mV
step without (solid triangles) and with (open triangles) 100 lm cAMP in the
pipette solution. For C, E and F control (n ¼ 9), control cAMP (n ¼ 8), MD
(n ¼ 12), MD cAMP (n ¼ 10), NH (n ¼ 10), NH cAMP (n ¼ 9). Data represent
means ± SEM. Throughout the figure: *P < 0.05 when compared to control
without cAMP; #P < 0.05 when compared to control with 100 lm cAMP in the
pipette solution.
permanent presence of 4 mm ATP in the pipette solution also excludes
any contribution of adenylate cyclase independent ATP effect to the
differences in Ih (Komagiri & Kitamura, 2004).
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 23, 3346–3358
Environment alters absence seizures, Ih, and HCN1 3353
Neonatal rearing conditions did not appear to affect the
membrane area, as estimated by whole cell capacitance (CM)
measurements (Table 2). There were no significant differences in
CM between normally reared WAG ⁄ Rij rats and MD or NH
(P > 0.1, respectively). Furthermore, rearing conditions did not
influence the relative contribution of different ionic permeabilities,
as assessed by reversal potential estimations (see Materials and
methods, Table 2).
HCN1 protein, but not mRNA expression is increased in
neocortex of NH and MD rats
To investigate whether the observed increase in Ih after NH and MD
might be accompanied by putative changes in the molecular correlates
of Ih, we performed in situ hybridization and Western blot to analyse
the distribution and amount of the channel subunits HCN1, 2 and 4.
Our Western blot analysis of HCN1 expression showed a marked
difference between the three groups (F2,6 ¼ 34.15, P ¼ 0.001). The
Scheffe posthoc test revealed an increased level of the HCN1 protein
in the neocortex of MD (833.33 ± 44.09 SEM, P < 0.05) and NH
(1233.33 ± 71.26 SEM, P ¼ 0.001) WAG ⁄ Rij rats, compared to
normal reared control WAG ⁄ Rij rats (556.67 ± 56.08 SEM). Further-
more, animals that underwent NH also had a higher HCN1 protein
expression compared to MD treated rats (P ¼ 0.008) (Fig. 6A).
Because the increase in HCN1 protein from control in MD and NH
rats paralleled the increase in Ih, or decrease in seizure activity,
respectively, we performed Pearson correlation analyses to investigate
further possible relationships between the three variables. The
analyses revealed that increases in HCN1 protein were highly
correlated with increases in Ih (r ¼ 0.91, P ¼ 0.13), suggesting that
current changes are mainly mediated by changes in this subunit.
Interestingly, we also found strong negative correlations between
HCN1 protein and seizure activity (r ¼ –0.94, P ¼ 0.11), and Ih and
seizure activity (r ¼ –0.99, P ¼ 0.02). These correlations provide
support that decreased seizure activity after MD or NH is associated
with increased neocortical Ih and HCN1.
In contrast to HCN1, Western blot analyses of the HCN2
(855 ± 64.07 SEM, control; 674.00 ± 94.02 SEM, MD;
889.67 ± 63.71 SEM, NH; F2,6 ¼ 2.37, P ¼ 0.18) and HCN4
(382.67 ± 6.77 SEM, control; 436.33 ± 51.17 SEM, MD;
341.67 ± 20.25 SEM, NH; F2,6 ¼ 2.2, P ¼ 0.19) subunits showed a
similar protein expression across all three neonatal rearing environ-
ment groups as depicted in Fig. 6C. We found no correlations between
HCN2 or 4 subunits and Ih or seizure activity, respectively.
Furthermore, although the mRNA signal of all three HCN subunits
in the neocortex of WAG ⁄ Rij rats showed the same distribution as
described previously (Strauss et al., 2004), we did not find any
differences in the amount of mRNA in any of the HCN subunits
between our three neonatal rearing environment groups (Fig. 6B). This
indicates that our observed increase in HCN1 in MD and NH
WAG ⁄ Rij rats compared to control animals is selective for the protein
level only.
Discussion
Our results show for the first time that environmental manipulations
early in development can influence absence seizures, Ih and HCN1
expression in WAG ⁄ Rij rats later in life. In particular, we found that
either neonatal handling or maternal deprivation during the first three
weeks of life led in adulthood to: (i) a pronounced seizure reduction,
accompanied by (ii) an increased Ih, (iii) faster activation ⁄ deactivation
3354 U. Schridde et al.

Table 2. Common voltage-clamp and Ih properties of layer 5 pyramidal neurons from somatosensory cortex of WAG ⁄ Rij rats raised under different rearing
conditions

Control (n) MD (n) NH (n)

C (pF) 109.0 ± 13.5 17 101.3 ± 5.4 22 122.0 ± 10.0 19

Vh-reversal (mV) )45.0 ± 1.5 5 )41.2 ± 1.3 8 )41.3 ± 1.9 6
V1 ⁄ 2 (mV) )85.3 ± 2.3 9 )86.4 ± 2.0 12 )86.1 ± 1.1 10
Slope-factor (k) 11.1 ± 0.8 9 10.7 ± 0.6 12 12.4 ± 0.4 10
V1 ⁄ 2 – 100 lm cAMP (mV) )82.6 ± 1.1 8 )85.6 ± 1.3 10 )84.8 ± 0.9 9
Slope-factor (k) – 100 lm cAMP 8.9 ± 0.8* 8 9.4 ± 0.2* 10 9.6 ± 0.4* 9

Data presented as the mean ± SEM (n, number of cells), *P < 0.05 when compared to slope-factors in the same group without cAMP.

kinetics of Ih, and (iv) an increased HCN1 expression, compared to
normally reared WAG ⁄ Rij rats.
Neonatal environmental manipulations reduce absence
seizures in adult WAG ⁄ Rij rats
We found that NH or MD resulted in less SWD than observed in
normal reared control WAG ⁄ Rij rats, while the mean duration of SWD
did not differ between groups. Our observation that environmental
manipulations influence number and mean duration of SWD differ-
ently is in line with previous research (Schridde & van Luijtelaar,
2004a). Different sensitivities of the various characteristics of SWD
were also reported for drugs (van Luijtelaar & Coenen, 1995; Schridde
& van Luijtelaar, 2004b); and evidence suggests that various SWDs or
their characteristics are influenced by different genes (Peeters et al.,
1990, 1992; Gauguier et al., 2004; Rudolf et al., 2004).
Neonatal handling and MD can influence an animals’ behaviour and
emotionality (Caldji et al., 2000). SWD are closely related to
behaviour and vigilance levels, being prominent during states of low
activity and drowsiness, but nearly absent during activity and arousal
(Coenen et al., 1991; Drinkenburg et al., 1991). However, the
similarity in behaviour between groups suggests that the decrease of
absence seizures in NH or MD reared rats is not related to changes in
behavioural states and hence might be ascribed to neuronal mecha-
nisms directly related to SWD. No differences between NH, MD and
normal reared control WAG ⁄ Rij rats in mainly stress-related behav-
iours were observed in our laboratory earlier (unpublished observa-
tion).
The reduction in absence seizures is correlated to increased
neocortical Ih and HCN1
Along with the seizure reduction in NH or MD WAG ⁄ Rij rats, whole-
cell recordings from layer 5 pyramidal neurons in the somatosensory
cortex of NH and MD rats revealed an increased (nearly doubled) Ih
density and faster activation ⁄ deactivation kinetics, compared to
normal reared control rats. Several lines of evidence suggest that
these changes represent larger channel availability due to a selective
increase in the HCN1 subunit (Franz et al., 2000), known to be widely
expressed in neocortical layer 5 pyramidal cells (Santoro et al., 2000),
in NH and MD WAG ⁄ Rij rats. First, the absence of differences in cell
capacitance between our three rearing environment groups under-
scores that the increase in current is not due to changes in cell size, this
is also reflected by the differences in the Ih densities. Second, Ih of NH
and MD rats differed from control in the rate of channel activation,
with Ih of NH and MD rats showing faster activation ⁄ deactivation
kinetics. It is generally agreed upon that the HCN1 subunit mediates
rapid activation of Ih, whereas HCN2, also prominent in the cortex,
shows slower kinetics (Santoro & Tibbs, 1999; Santoro et al., 2000).
Third, our observation that V½ and the slope-factor of the voltage
sensitivity (k) did not differ between control, NH and MD rats, even
after the application of cAMP, makes it very unlikely that the group
differences in Ih were caused by changes in basal levels of underlying
cyclic nucleotides. The finding that the application of cAMP neither
affected the group differences in Ih amplitude and density, nor the rate
of activation ⁄ deactivation, and only slightly shifted the V½, supports
again a selective involvement of the less cAMP sensitive HCN1
(Robinson & Siegelbaum, 2003). Finally, in line with the aforemen-
tioned findings, our Western blot analyses revealed a selective
up-regulation of the HCN1 subunit at the somatosensory cortex of
NH and MD WAG ⁄ Rij rats compared to control, while HCN2 and 4
were not different between groups.
Putative mechanisms of Ih and HCN1 modification
Our data revealed that HCN1 changed only at the protein level, but not
at the level of mRNA. Interestingly, differences in HCN1 between
epileptic WAG ⁄ Rij and nonepileptic control strains reported earlier
were also selective for the protein only (Strauss et al., 2004). This
suggests that the increase in HCN1 in NH and MD rats might be due
to group differences in post-transcriptional or post-translational factors
that affect the amount or location of HCN1 proteins. Although
relatively little is known about such factors, current research points to
protein–protein interactions as a putative explanation as such a
mechanism would influence Ih in a way similar to our results and it
involves HCN channel expression, implementation, or localizing, in
some studies (Gravante et al., 2004; Vasilyev & Barish, 2004) with a
preference for the HCN1 subunit. In particular, the extracellular matrix
protein vitronectin increased the amplitude and accelerated the
activation of Ih in hippocampal pyramidal cells (especially the fast
component of Ih being sensitive for vitronectin), while not affecting its
voltage sensitivity. Immunofluorescence measures revealed that
vitronectin elevated HCN1 while leaving HCN2 unaffected (Vasilyev
& Barish, 2004). A second protein involved in channel localization
and functioning is the cytoplasmatic scaffold protein filamin A. In
filamin A deficient cells the normalized conductance mediated by
HCN1 was approximately two times larger and channel activa-
tion ⁄ deactivation were markedly accelerated. No interactions were
found between filamin A and the subunits HCN2 and HCN4
(Gravante et al., 2004). Thirdly, changes in Ih and HCN1 might arise
from an increase in the transmembrane-spanning protein MiRP1 that
acts as a b-subunit for the HCN channels. MiRP1 increased Ih and
speeded up its activation, as well as influenced the expression of the
HCN1 channel in Xenopus oocytes (Yu et al., 2001). Although not
restricted to HCN1 the TPR-containing Rab8b interacting protein
(TRIP8b) caused a specific decrease in the surface expression of HCN
protein and Ih density in expression systems, as well as in cultured
pyramidal neurons, again the voltage dependence being unaffected

ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 23, 3346–3358
 Environment alters absence seizures, Ih, and HCN1 3355

Fig. 6. Analysis of HCN1, 2 and 4 protein expression in the neocortex of rats from the three neonatal rearing environments. (A, left) Western blots of HCN1 from
total protein extracts of adult rats from the three neonatal rearing environments; WAG ⁄ Rij control, WAG ⁄ Rij maternal deprivation, and WAG ⁄ Rij neonatal handling.
Right, quantification of respective Western blot analyses per group (each n ¼ 3), indicating that rats that underwent maternal deprivation (WAG ⁄ Rij MD) or
neonatal handling (WAG ⁄ Rij NH) had an increased HCN1 protein expression when compared to control rats (WAG ⁄ Rij control); and WAG ⁄ Rij NH also had a
higher HCN1 protein expression as WAG ⁄ Rij MD rats. *Indicates significant difference between WAG ⁄ Rij control compared to WAG ⁄ Rij MD (P < 0.05) or
WAG ⁄ Rij NH (P ¼ 0.001), respectively; and also between WAG ⁄ Rij MD and WAG ⁄ Rij NH (P ¼ 0.008) as revealed by anova and Scheffe posthoc test.
(B) In situ hybridization analysis of HCN1 mRNA expression in the neocortex of rats from the three neonatal rearing environments. No alterations are detectable
between the three groups. (C) Western blots and quantitative analyses between the three neonatal rearing environments (each n ¼ 3) for HCN2 (left) and HCN4
(right) subunits, respectively. In contrast to HCN1, the HCN2 and HCN4 subunits showed a similar protein expression across all three neonatal rearing environment
groups. All data in histograms are displayed as mean ± SEM. Calibration bar, 1.125 mm.
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 23, 3346–3358
3356 U. Schridde et al.
(Santoro et al., 2004). Although it is not yet clear whether NH or MD
might influence any of the above-mentioned proteins specifically or in
combination, it is challenging to answer this question in future
research.
Functional consequences of an increased Ih
Ih plays a crucial role in the regulation of the resting membrane
potential and cellular excitability (Robinson & Siegelbaum, 2003) and
is closely linked to hyperexcitability during pathological conditions
such as epilepsy (Di Pasquale et al., 1997; Chen et al., 2001; Poolos
et al., 2002; Shah et al., 2004). Especially for WAG ⁄ Rij rats – a model
for absence epilepsies – a decrease in Ih in pyramidal cells of the
somatosensory cortex was recently associated with an increased
cellular excitability, thought to promote the generation of absence
seizures in WAG ⁄ Rij rats (Strauss et al., 2004). It is noteworthy that
NH or MD increased HCN1 protein in the somatosensory cortex of
WAG ⁄ Rij rats up to a level similar to that of Wistar rats reported by
Strauss et al. (2004). Interestingly, Wistar rats are not completely
devoid of SWD, but their seizures are much less prominent than in
WAG ⁄ Rij rats (Coenen & van Luijtelaar, 2003). Also in our EEG
recordings NH or MD WAG ⁄ Rij rats were not free of, but showed
considerably less seizures, compared to normally reared control
animals. Next, the order of the magnitude of our observed group
differences in seizure activity (NH ⁄ MD < Control) was the same,
although reversed, as in our group differences in Ih and HCN1
(NH ⁄ MD > Control). These observations suggest a negative relation-
ship between seizure activity and Ih ⁄ HCN1 (confirmed by negative
correlations), and support the role of Ih and HCN1 as one putative
mediator of decreased absence seizures in NH or MD reared WAG ⁄ Rij
rats.
Yet in how far the relationship between Ih ⁄ HCN1 and seizure
activity also reflects causality needs to be further investigated. In
particular a contribution of hypothalamic-pituitary-adrenal axis hor-
mones (e.g. corticosteroids) and ⁄ or GABAergic systems, both sensi-
tive for early environmental manipulations like NH and MD (Caldji
et al., 2000; Hsu et al., 2003; Pryce & Feldon, 2003) and influencing
SWD (Manning et al., 2003; Schridde & van Luijtelaar, 2004b),
cannot be ruled out. However, earlier we found no significant
differences in plasma levels of corticosterone between adult NH, MD
or normal reared control WAG ⁄ Rij rats (unpublished observation).
So far, available data on the association between epilepsy and
changes in Ih and HCN are controversial, however, showing either a
positive or a negative relationship between Ih ⁄ HCN and seizure
activity. Some reports argue for an increased Ih associated with
epilepsy, as for absence epilepsy in stargazer mice (Di Pasquale et al.,
1997), for temporal lope epilepsy patients (Bender et al., 2003) and for
febrile seizure models (Chen et al., 2001; Brewster et al., 2002),
corroborated by antiepileptic effects by a block of Ih (Kitayama et al.,
2003).
However, our data support the hypothesis of a decreased Ih in
epilepsy as shown for a genetic model of absence epilepsy (Strauss
et al., 2004), in the kainic acid temporal lobe epilepsy model (Shah
et al., 2004), and for HCN2 knock-out mice which develop absence
seizures (Ludwig et al., 2003). In the thalamocortical loop, a structure
involved in the pathogenesis of absence epilepsy, synchronized
rhythms are controlled by Ih and it has been suggested, that the
up-regulation of Ih might be a key player in the cessation of these
events (Lüthi & McCormick, 1998). In line with the latter it is
interesting that a recent study by Budde et al. (2005) showed an
impaired regulation of Ih in the thalamus of WAG ⁄ Rij rats compared
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
to nonepileptic ACI rats, thereby facilitating seizure generation.
Another hint for the protecting role of Ih in epilepsy is the observation
that drugs that reduce seizures increase Ih (Munsch & Pape, 1999;
Poolos et al., 2002; Surges et al., 2003).
The seemingly conflicting functions of Ih in epilepsy, i.e. pro- or anti-
excitatory effects of Ih, have been the topic of intense speculation. The
net impact of changes in Ih is likely to be determined by other ionic
conductances present in the respective neuron – in particular the
interplay between Ih and IT, by the nature and spatial distribution of
synaptic input and by biophysical properties, or by subunit composition
and subcellular localization of the h-channels, especially as the
molecular subunit composition itself might alter the localization
(reviewed in Santoro & Baram, 2003). In addition all these determinants
might vary in the specific epilepsy models. Nevertheless, evidence is
accumulating for a putative ‘h-channelopathy’ (Poolos, 2005) in
epilepsy. By showing that Ih and HCN1 can be changed by environ-
mental manipulations early in life, accompanied by a reduction of
absence seizures, our results make an important contribution to this field.
In conclusion, our observations provide the first electrophysiolog-
ical (in vivo and in vitro) and molecular biological evidence that an
increase of Ih and HCN1 is associated with absence seizure reduction.
While in the literature environmental factors that influence seizures
usually refer to major insults, we could show that in WAG ⁄ Rij rats
absence seizures, Ih and HCN1 can also be permanently influenced by
relatively mild changes in the neonatal environment. Our observations
may lead to an increase in the knowledge of environmental influences
on Ih and HCN, and their role in the normal and deviant development
and functioning of the nervous system. In particular environmental
influences on Ih and HCN in the normal brain, and its consequences
for cortical functioning, would be an interesting topic for future
studies. In the case of epilepsy our findings demonstrate that
genetically determined absence seizures are quite sensitive for early
interventions.
Acknowledgements
The valuable help of the following people is kindly acknowledged: Hans
Krijnen and Saskia Hermeling for animal caretaking, Gerard van Oijen for
general assistance and Carla Biedermann for technical assistance in electro-
physiological experiments, Bettina Brokowski for technical assistance in
Western blot analysis, and Dr Hal Blumenfeld for critical reading of the
manuscript and valuable comments. Ulrich Schridde and Gilles van Luijtelaar
had been supported by a NWO grant (425-20-401), Ulf Strauss had been
partially supported by a GSK grant for epilepsy research.
Abbreviations
ACSF, artificial cerebrospinal fluid; HCN, hyperpolarization-activated cation
channel; ICa, Ca2+ current; Ih, hyperpolarization-activated cation current; IR-DIC,
infrared differential interference contrast; IT, low-threshold Ca2+ current; MD,
maternal deprivation; NH, neonatal handling; SWD, spike-wave discharges;
TTX, tetrodotoxin; WAG ⁄ Rij, Wistar Albino Glaxo ⁄ Rijswijk rat; ZD7288,
4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride.
References
Andersen, S.L. (2003) Trajectories of brain development: point of
vulnerability or window of opportunity? Neurosci. Biobehav. Rev., 27,
3–18.
Bender, R.A., Brewster, A., Santoro, B., Ludwig, A., Hofmann, F., Biel, M. &
Baram, T.Z. (2001) Differential and age-dependent expression of hyperpo-
larization-activated, cyclic nucleotide-gated cation channel isoforms 1–4
suggests evolving roles in the developing rat hippocampus. Neuroscience,
106, 689–698.
European Journal of Neuroscience, 23, 3346–3358

Bender, R.A., Soleymani, S.V., Brewster, A.L., Nguyen, S.T., Beck, H.,
Mathern, G.W. & Baram, T.Z. (2003) Enhanced expression of a specific
hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN) in
surviving dentate gyrus granule cells of human and experimental epileptic
hippocampus. J. Neurosci., 23, 6826–6836.
Bräuer, A.U., Savaskan, N.E., Kole, M.H.P., Plaschke, M., Monteggia, L.M.,
Nestler, E.J., Simburger, E., Deisz, R.A., Ninnemann, O. & Nitsch, R.
(2001a) Molecular and functional analysis of hyperpolarization-activated
pacemaker channels in the hippocampus after entorhinal cortex lesion.
FASEB J., 15, 2689–2701.
Bräuer, A.U., Savaskan, N.E., Plaschke, M., Ninnemann, O. & Nitsch, R.
(2001b) Perforant path lesion induces up-regulation of stathmin messenger
RNA, but not SCG10 messenger RNA, in the adult rat hippocampus.
Neuroscience, 102, 515–526.
Brewster, A., Bender, R.A., Chen, Y., Dube, C., Eghbal-Ahmadi, M. & Baram,
T.Z. (2002) Developmental febrile seizures modulate hippocampal gene
expression of hyperpolarization-activated channels in an isoform- and cell-
specific manner. J. Neurosci., 22, 4591–4599.
Budde, T., Caputi, L., Kanyshkova, T., Staak, R., Abrahamczik, C., Munsch, T.
& Pape, H.C. (2005) Impaired regulation of thalamic pacemaker channels
through an imbalance of subunit expression in absence epilepsy. J. Neurosci.,
25, 9871–9882.
Caldji, C., Francis, D., Sharma, S., Plotsky, P.M. & Meaney, M.J. (2000) The
effects of early rearing environment on the development of GABAA and
central benzodiazepine receptor levels and novelty-induced fearfulness in the
rat. Neuropsychopharmacology, 22, 219–229.
Chen, K., Aradi, I., Santhakumar, V. & Soltesz, I. (2002) H-channels in
epilepsy: new targets for seizure control? TIPS, 23, 552–557.
Chen, K., Aradi, I., Thon, N., Eghbal-Ahmadi, M., Baram, T.Z. & Soltez, I.
(2001) Persistently modified h-channels after complex febrile seizures
convert the seizure-induced enhancement of inhibition to hyperexcitability.
Nature Med., 7, 331–337.
Coenen, A.M.L., Drinkenburg, W.H.I.M., Peeters, B.W.M.M., Vossen, J.M.H.
& van Luijtelaar, E.L.J.M. (1991) Absence epilepsy and the level of
vigilance in rats of the WAG ⁄ Rij strain. Neurosci. Biobehav. Rev., 15, 259–
263.
Coenen, A.M.L. & van Luijtelaar, E.L.J.M. (2003) Genetic animal models for
absence epilepsy: a review of the WAG ⁄ Rij strain of rats. Behav. Genet., 33,
635–655.
Crunelli, V. & Leresche, N. (2002) Childhood absence epilepsy: genes,
channels, neurons and networks. Nature Rev. Neurosci., 3, 371–382.
Di Pasquale, E., Keegan, K.D. & Noebels, J.L. (1997) Increased excitability
and inward rectification in layer V cortical pyramidal neurons in the epileptic
mutant stargazer. J. Neurophysiol., 77, 621–631.
Drinkenburg, W.H.I.M., Coenen, A.M.L., Vossen, J.M.H. & van Luijtelaar,
E.L.J.M. (1991) Spike-wave discharges and sleep-wake states in rats with
absence epilepsy. Epilepsy Res., 9, 218–224.
Franz, O., Liss, B., Neu, A. & Roeper, J. (2000) Single-cell mRNA expression
of HCN1 correlates with a fast gating phenotype of hyperpolarization-
activated cyclic nucleotide-gated ion channels (Ih) in central neurons. Eur. J.
Neurosci., 12, 2685–2693.
Gauguier, D., van Luijtelaar, G., Bihoreau, M.T., Wilder, S.P., Godfrey, R.F.,
Vossen, J., Coenen, A. & Cox, R.D. (2004) Chromosomal mapping of
genetic loci controlling absence epilepsy phenotypes in the WAG ⁄ Rij rat.
Epilepsia, 45, 908–915.
Gravante, B., Barbuti, A., Milanesi, R., Zappi, I., Viscomi, C. & DiFrancesco,
D. (2004) Interaction of the pacemaker channel HCN1 with filamin A.
J. Biol. Chem., 279, 43847–43853.
Harris, N.C. & Constanti, A. (1995) Mechanism of block by ZD 7288 of the
hyperpolarization-activated inward rectifying current in guinea pig substantia
nigra neurons in vitro. J. Neurophysiol., 74, 2366–2378.
Hsu, F.C., Zhang, G.J., Raol, Y.S.H., Valentino, R.J., Coulter, D.A. &
Brooks-Kayal, A.R. (2003) Repeated neonatal handling with maternal
separation permanently alters hippocampal GABAA receptors and
behavioral stress responses. Proc. Natl Acad. Sci. USA, 100, 12213–
12218.
Kitayama, M., Miyata, H., Yano, M., Saito, N., Matsuda, Y., Yamauchi, T. &
Kogure, S. (2003) Ih blockers have a potential antiepileptic effects.
Epilepsia, 44, 20–24.
Komagiri, Y. & Kitamura, N. (2004) Effect of intracellular dialysis of ATP on
the hyperpolarization-activated cation current in rat dorsal root ganglion
neurons. J. Neurophysiol., 90, 2115–2122.
Lorincz, A., Notomi, T., Tamas, G., Shigemoto, R. & Nusser, Z. (2002)
Polarized and compartment-dependent distribution of HCN1 in pyramidal
cell dendrites. Nature Neurosci., 5, 1185–1193.
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 23, 3346–3358
Environment alters absence seizures, Ih, and HCN1 3357
Ludwig, A., Budde, T., Stieber, J., Moosmang, S., Wahl, C., Holthoff, K.,
Langebartels, A., Wotjak, C., Munsch, T., Zong, X., Feil, S., Feil, R., Lancel,
M., Chien, K.R., Konnerth, A., Pape, H.C., Biel, M. & Hofmann, F. (2003)
Absence epilepsy and sinus dysrhythmia in mice lacking the pacemaker
channel HCN2. EMBO J., 22, 216–224.
Ludwig, A., Zong, X., Jeglitsch, M., Hofmann, F. & Biel, M. (1998) A family
of hyperpolarization-activated mammalian cation channels. Nature, 393,
587–591.
Lüthi, A. & McCormick, D.A. (1998) Periodicity of thalamic oscillations: the
role of Ca2+-mediated upregultion of Ih. Neuron, 20, 553–563.
Manning, J.P., Richards, D.A. & Bowery, N.G. (2003) Pharmacology of
absence epilepsy. TIPS, 24, 542–549.
Manning, J.P.A., Richards, D.A., Leresche, N., Crunelli, V. & Bowery, N.G.
(2004) Cortical-area specific block of genetically determined absence
seizures by ethosuximide. Neuroscience, 123, 5–9.
Meeren, H.K.M., Pijn, J.P.M., van Luijtelaar, E.L.J.M., Coenen, A.M.L. &
Lopes da Silva, F.H. (2002) Cortical focus drives widespread corticothalamic
networks during spontaneous absence seizures in rats. J. Neurosci., 22,
1480–1495.
Munsch, T. & Pape, H.C. (1999) Upregulation of the hyperpolarization-
activated cation current in rat thalamic relay neurons by acetazolamide.
J. Physiol. (Lond.), 519, 505–514.
Noldus, L.P.J.J. (1991) The observer: a software system for collecting and
analysis of observational data. Behav. Res. Meth. Instrum. Comput., 23, 415–
429.
Paxinos, G. & Watson, C. (1998) The Rat Brain in Stereotaxic Coordinates, 4th
Edn. Academic Press, San Diego.
Peeters, B.W.M.M., Kerbusch, J.M.L., Coenen, A.M.L., Vossen, J.M.H. & van
Luijtelaar, E.L.J.M. (1992) Genetics of spike-wave discharges in the
electroencephalogram (EEG) of the WAG ⁄ Rij inbred rat strain: a classical
mendelian crossbreeding study. Behav. Genet., 22, 361–368.
Peeters, B.W.M.M., Kerbusch, J.M.L., van Luijtelaar, E.L.J.M., Vossen, J.M.H.
& Coenen, A.M.L. (1990) Genetics of absence epilepsy in rats. Behav.
Genet., 20, 453–460.
Poolos, N.P. (2004) The Yin and Yang of the H-channel and its role in epilepsy.
Epilepsy Currents, 4, 3–6.
Poolos, N.P. (2005) The h-channel: a potential channelopathy in epilepsy?
Epilepsy Behav., 7, 51–56.
Poolos, N., Migliore, M. & Johnson, D. (2002) Pharmacological upregulation
of h-channels reduces the excitability of pyramidal neuron dendrites. Nature
Neurosci., 5, 767–774.
Pryce, C.R. & Feldon, J. (2003) Long-term neurobehavioural impact of the
postnatal environment in rats: manipulations, effects and mediating
mechanisms. Neurosci. Biobehav. Rev., 27, 57–71.
Roberts, L. & Greene, J.R.T. (2005) Hyperpolarization-activated current (Ih): a
characterization of subicular neurons in brain slices from socially and
individually housed rats. Brain Res., 1040, 1–13.
Robinson, R.B. & Siegelbaum, S.A. (2003) Hyperpolarization-activated cation
currents: from molecules to physiological function. Annu. Rev. Physiol., 65,
453–480.
Rudolf, G., Bihoreau, M.T., Godfrey, R., Cox, R.D., Lathrop, M., Marescaux,
C. & Gauguier, D. (2004) Polygenetic control of idiopathic generalized
epilepsy phenotypes in the Genetic Absence Rats from Strasbourg (GAERS).
Epilepsia, 45, 301–308.
Santoro, B. & Baram, T.Z. (2003) The multiple personalities of h-channels.
TINS, 10, 550–554.
Santoro, B., Chen, S., Lüthi, A., Pavlidis, P., Shumyatsky, G.P., Tibbs, G.R. &
Siegelbaum, S.A. (2000) Molecular and functional heterogeneity of
hyperpolarization-activated pacemaker channels in the mouse CNS.
J. Neurosci., 20, 5264–5275.
Santoro, B., Grant, S.G., Bartsch, D. & Kandel, E.R. (1997) Interactive cloning
with the SH3 domain of N-src identifies a new brain specific ion channel
protein, with homology to eag and cyclic nucleotide-gated channels. Proc.
Natl Acad. Sci. USA, 94, 14815–14820.
Santoro, B. & Tibbs, G.R. (1999) The HCN gene family: molecular basis of the
hyperpolarization-activated pacemaker channels. Ann. N.Y. Acad. Sci., 868,
741–764.
Santoro, B., Wainger, B.J. & Siegelbaum, S.A. (2004) Regulation of HCN
channel surface expression by a novel C-terminal protein–protein interaction.
J. Neurosci., 24, 10750–10762.
Schridde, U. & van Luijtelaar, G. (2004a) The influence of strain and housing
on two types of spike-wave discharges in rats. Genes, Brain Behav., 3, 1–7.
Schridde, U. & van Luijtelaar, G. (2004b) Corticosterone increases spike-wave
discharges in a dose and time dependent manner in WAG ⁄ Rij rats.
Pharmacol. Biochem. Behav., 78, 369–375.
3358 U. Schridde et al.
Sciancalepore, M. & Constanti, A. (1998) Inward-rectifying membrane currents
activated by hyperpolarization in immature rat olfactory cortex neurones
in vitro. Brain Res., 814, 133–142.
Shah, M.M., Anderson, A.E., Leung, V., Lin, X. & Johnston, D. (2004)
Seizure-induced plasticity of h channels in entorhinal cortical layer III
pyramidal neurons. Neuron, 44, 495–508.
Strauss, U., Kole, M.H.P., Bräuer, A.U., Pahnke, J., Bajorat, R., Rolfs, A.,
Nitsch, R. & Deisz, R.A. (2004) An impaired neocortical Ih is associated
with enhanced excitability and absence epilepsy. Eur. J. Neurosci., 19, 3048–
3058.
Surges, R., Freiman, T.M. & Feuerstein, T.J. (2003) Gabapentin increases the
hyperpolarization-activated cation current Ih in rat CA1 pyramidal cells.
Epilepsia, 44, 150–156.
van Luijtelaar, E.L.J.M. & Coenen, A.M.L. (1986) Two types of electrocortical
paroxysms in an inbred strain of rats. Neurosci. Lett., 70, 393–397.
van Luijtelaar, E.L.J.M. & Coenen, A.M.L. (1988) Circadian rhythmicity in
absence epilepsy in rats. Epilepsy Res., 2, 331–336.
van Luijtelaar, E.L.J.M. & Coenen, A.M.L. (1995) Effects of remacemide and
its metabolite FPL 12495 on spike-wave discharges in rats with absence
epilepsy. Neuropharmacology, 34, 419–425.
van Luijtelaar, E.L.J.M., Dirksen, R., Vree, T. & van Haaren, F. (1996) Effects
of acute and chronic cocaine administration on EEG and behaviour in intact
ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
and castrated male and intact and ovariectomized female rats. Brain Res.
Bull., 40, 43–50.
Vasilyev, D.V. & Barish, M.E. (2002) Postnatal development of the
hyperpolarization-activated excitatory current Ih in mouse hippocampal
pyramidal neurons. J. Neurosci., 22, 8992–9004.
Vasilyev, D.V. & Barish, M.E. (2004) Regulation of the hyperpolarization-
activated cationic current Ih in mouse hippocampal pyramidal neurons by
vitronectin, a component of extracellular matrix. J. Physiol., 560, 659–
675.
Wainger, B.J., DeGennaro, M., Santoro, B., Siegelbaum, S.A. & Tibbs, G.R.
(2001) Molecular mechanisms of cAMP modulation of HCN pacemaker
channels. Nature, 411, 805–810.
Weizman, R., Lehmann, J., Leschiner, S., Allmann, I., Stoehr, T., Heidbreder,
C., Domeney, A., Feldon, J. & Gavish, M. (1999) Long-lasting effect of early
handling on the peripheral benzodiazepine receptor. Pharmacol. Biochem.
Behav., 64, 725–729.
Yu, H., Wu, J., Potapova, I., Wymore, R.T., Holmes, B., Zuckerman, J.,
Pan, Z., Wang, H., Shi, W., Robinson, R.B., El-Maghrabi, M.R.,
Benjamin, W., Dixon, J., McKinnon, D., Cohen, I.S. & Wymore, R.
(2001) MinK-related peptide 1: A b subunit for the HCN ion channel
subunit family enhances expression and speeds activation. Circ. Res., 88,
e84–e87.
European Journal of Neuroscience, 23, 3346–3358