Epilepsy Research 145 (2018) 63–72

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Epilepsy Research
journal homepage: www.elsevier.com/locate/epilepsyres

Carbamazepine-induced suppression of repetitive firing in CA1 pyramidal T

neurons is greater in the dorsal hippocampus than the ventral hippocampus
⁎
Madeline Collette Evans, Kelly Ann Dougherty
Department of Biology, Rhodes College, 2000 N Parkway, Memphis, TN, 38112, USA

A R T I C LE I N FO A B S T R A C T

Keywords: Medial temporal lobe epilepsy (mTLE)–the most common form of focal epilepsy–is defined by recurrent partial
Carbamazepine seizures originating within the medial temporal lobe. Such seizures are commonly associated with the anterior
Dorsal hippocampus hippocampus (as opposed to the posterior hippocampus), and refractory to the currently available anti-epileptic
Intrinsic excitability drugs (AED) for about one third of patients. Unfortunately, the mechanisms driving seizure generation and AED
Anti-Epileptic drug
efficacy along the longitudinal hippocampal axis remain poorly understood. Recently, several groups in-
CA1 pyramidal neuron
vestigating differences in excitability along the rodent longitudinal hippocampal axis have demonstrated that
CA1 pyramidal neurons from the rodent ventral hippocampus (the rodent homolog of the human anterior
hippocampus) are intrinsically more excitable than their dorsal counterparts (the rodent homolog of the human
posterior hippocampus). This phenotypic difference is accompanied by significant differences in gene expression
along the longitudinal hippocampal axis, which include gene products–such as voltage-gated sodium channel β-
subunits–known to influence AED efficacy. Given this phenotypic heterogeneity, and the differential expression
of gene products known to influence anti-epileptic drug efficacy, we sought to investigate the efficacy of the
classical use-dependent sodium channel blocker, carbamazepine, in CA1 pyramidal neurons across the long-
itudinal hippocampal axis. Accordingly, we performed whole-cell current-clamp recordings on CA1 pyramidal
neurons from acute hippocampal slices prepared from the dorsal and ventral hippocampus, and found that acute
exposure to 100 μM carbamazepine induced a significantly greater suppression of repetitive firing for dorsal
neurons relative to ventral neurons by inducing profound spike frequency adaptation (SFA). Moreover, we
observed a small, but significant depolarization of resting membrane potential (RMP) for dorsal neurons (but not
ventral neurons), following exposure to carbamazepine. Together, these observations demonstrate that carba-
mazepine’s effect is concentrated in the dorsal hippocampus, which could provide meaningful insight into the
side effect profile of carbamazepine (and related anti-epileptic drugs) in non-epileptic tissue, and inform future
work investigating the mechanisms of carbamazepine resistance in epileptic tissue.

1. Introduction seizures in about one-third of mTLE patients, thereby necessitating the

Medial temporal lobe epilepsy (mTLE)–the most common form of
focal epilepsy–is defined by partial seizures that spontaneously origi-
nate within the medial temporal lobe. Specifically, seizures often ori-
ginate in the anterior region of the hippocampal formation before
spreading to other regions of the hippocampus and brain, which is as-
sociated with cell death and hippocampal sclerosis within the anterior
hippocampus (Engel et al., 1990; Babb et al., 1984). Unfortunately,
currently available anti-epileptic drugs (AED) do not adequately control
surgical resection of the anterior hippocampus (Kwan and Sander,
2004). Although the specific mechanisms driving the hyperexcitability
of the human anterior hippocampus remain poorly understood, recent
work exploring the rodent ventral hippocampus (the rodent homolog of
the human anterior hippocampus) has begun to yield insight into the
special excitability of this brain region (Dougherty et al., 2012;
Hönigsperger et al., 2015; Marcelin et al., 2012; Kim and Johnston,
2015; Malik and Johnston, 2017). Accordingly, several groups have
identified significant heterogeneity in anatomy, synaptic connectivity,

Abbreviations: mTLE, medial temporal lobe epilepsy; AED, anti-epileptic drug; DHC, dorsal hippocampus; dIHC, dorsal intermediate hippocampus; vIHC, ventral intermediate hip-
pocampus; VHC, ventral hippocampus; ISA, somatodendritic A-type current; IM, m-current; GIRK, channel, G-protein coupled inwardly recitifying potassium channel; CBZ, carbamazepine;
aCSF, artificial cerebrospinal fluid; DNQX, 6,7-dinitroquinoxaline 2,3-dione; DMSO, dimethyl sulfoxide; Rin, input resistance; τM, membrane time constant; Vthr, threshold voltage; APPeak,
action potential peak; APsize, action potential size; APhw, action potential half width; ISI, interspike interval; SFA, spike frequency adaptation; PTSD, post-traumatic stress disorder; FPKM,
fragments per kilobase of exon per million fragments mapped
⁎
Corresponding author at: 2000 N Parkway, Memphis, TN, 38112, USA.
E-mail address: doughertyk@rhodes.edu (K.A. Dougherty).

https://doi.org/10.1016/j.eplepsyres.2018.05.014
Received 7 March 2018; Received in revised form 11 May 2018; Accepted 29 May 2018
Available online 09 June 2018
0920-1211/ © 2018 Elsevier B.V. All rights reserved.
M.C. Evans, K.A. Dougherty
network-level phenomena, cellular morphology, cellular physiology,
and gene expression along the longitudinal hippocampal axis (dor-
sal–ventral axis; (Strange et al., 2014). An emerging theme from this
work is that the trisynaptic circuits of the dorsal hippocampus (DHC)
and ventral hippocampus (VHC) show quantitative differences, despite
their qualitative similarity. This “tuning” of the trisynaptic circuit likely
underlies the hyperexcitability of the VHC, when compared with the
DHC.
Recently, a more nuanced picture of these dorsal and ventral phe-
notypes has begun to emerge, as several groups have reported sig-
nificant physiological differences between CA1 pyramidal neurons from
the dorsal and ventral hippocampus. Dorsal CA1 pyramidal neurons are
less intrinsically excitable than their ventral counterparts, due to the
increased activity of A-type potassium channels underlying the soma-
todendritic A-type potassium current (ISA), the increased activity of
KCNQ channels underlying the m-current (IM), and the hyperpolariza-
tion of resting membrane potential (RMP) stemming from decreased
activity of the hyperpolarization-activated cation non-selective current
(Ih) (Dougherty et al., 2012, 2013; Marcelin et al., 2012; Hönigsperger
et al., 2015; Milior et al., 2016). Additionally, the expression of GIRK
channels coupled with A1 adenosine receptors further suppresses the
intrinsic excitability of DHC neurons, thereby limiting the ability of
these neurons to associate synaptic inputs over relatively long periods
of time (Kim and Johnston, 2015; Malik and Johnston, 2017). These
contrasting DHC and VHC phenotypes gradually blend together through
the intermediate hippocampus (IHC), as the intrinsic excitability of
individual neurons from intermediate positions along the longitudinal
hippocampal axis (i.e., the dorsal intermediate hippocampus, or dIHC,
and the ventral intermediate hippocampus, or vIHC) lies between the
hypoexcitable DHC phenotype and the hyperexcitable VHC phenotype
(Malik et al., 2016). This phenotypic transition along the longitudinal
hippocampal axis is supported by several studies describing discrete
dorsal, intermediate, and ventral CA1 regions along the longitudinal
hippocampal axis defined by changes in gene expression (Dong et al.,
2009; Fanselow and Dong, 2010; Cembrowski et al., 2016a). Among
these region-specific genes are several determinants of intrinsic excit-
ability, which include, but are not limited to voltage-gated ion channels
and their auxiliary subunits. Interestingly, one recent study by
Cembrowski et al. (2016a) demonstrated a significant enrichment of the
sodium channel β4 subunit (Scn4b) in CA1 pyramidal neurons from the
DHC, thereby implying differences in the α–β subunit composition of
voltage-gated sodium channels in these neurons (Cembrowski et al.,
2016a). Using a publically searchable hippocampal transcriptome da-
tabase (HippoSeq; http://hipposeq.janelia.org/), we examined the ex-
pression profiles of the remaining sodium channel subunits across the
longitudinal hippocampal axis, and found that three of the four sodium
channel β-subunits (β1, β3, and β4) showed increased expression in the
DHC relative to the VHC, whereas the β2 subunit was expressed simi-
larly across the longitudinal hippocampal axis (Table 1; Cembrowski
et al., 2016b).
Given the physiological and molecular heterogeneity of CA1 pyr-
amidal neurons along the longitudinal hippocampal axis, the increased
expression of sodium channel β-subunits in the DHC, and the differing
roles of the DHC and VHC in seizure generation, we hypothesized that
the classical anti-epileptic drug, carbamazepine (CBZ), which limits
excitability through a use-dependent sodium channel blocking me-
chanism, would differentially influence intrinsic excitability across the
longitudinal hippocampal axis. Accordingly, we found that acute ex-
posure to 100 μM CBZ 1) induces a greater suppression of repetitive
firing in the DHC than the VHC, and 2) results in a significant depo-
larization of RMP for DHC neurons, but not VHC neurons. These find-
ings underscore the molecular and physiological heterogeneity of CA1
neurons across the longitudinal hippocampal axis, suggest a unique role
for the DHC in the side effect profile of carbamazepine in non-epileptic
tissue, and prompt compelling questions concerning carbamazepine’s
efficacy along the longitudinal hippocampal axis in epileptic tissue.
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Epilepsy Research 145 (2018) 63–72
Table 1
Expression of neuronal voltage-gated sodium channel α- and β-subunits across
the longitudinal hippocampal axis.
Sodium Channel HippoSeq Gene CA1 DHC (FPKM) CA1 VHC (FPKM)
or β-subunit ID
Nav1.1 Scn1a 46.8 ± 24.5 33.3 ± 20.6
Nav1.2 Scn2a1 111.5 ± 41.5 136.2 ± 45.0
Nav1.3 Scn3a 11.6 ± 4.2 10.1 ± 4.2
Nav1.6 Scn8a 104.8 ± 22.2 84.2 ± 18.3
β1 Scn1b 81.7 ± 25.8a 38.8 ± 15.4a
β2 Scn2b 56.9 ± 15.2 44.1 ± 12.7
β3 Scn3b 322.7 ± 75.4b 85.8 ± 26.4b
β4 Scn4b 10.4 ± 4.3c 0.0 ± 0.0c
Relative transcript abundance was determined using HippoSeq, a publically
searchable hippocampal transcriptome database (http://hipposeq.janelia.org/;
Cembrowski et al., 2016b). Transcript abundance is presented as the average of
at least three replicates expressed in fragments per kilobase of exon per million
fragments mapped (FPKM) bounded by their 95% confidence interval. Super-
scripted letters indicate dorsal–ventral pairs with non-overlapping 95% con-
fidence intervals.
2. Materials and methods
2.1. Acute hippocampal slice preparation
Acute hippocampal slices were prepared from 22 adult male
Sprague-Dawley rats (4.7 ± 0.5 months; 403 ± 12 g) in accordance
withe the rules and regulations of the Rhodes College Institutional
Animal Care and Use Committee. Rats were deeply anesthetized with a
mixture of ketamine and xylazine immediately prior to transcardial
perfusion with a cutting saline of the following composition (in mM):
210 sucrose, 1.25 NaH2PO4, 25 NaHCO3, 2.5 KCl, 0.5 CaCl2, 7 MgCl2, 7
dextrose, 1.3 ascorbic acid, and 3 sodium pyruvate (continuously
bubbled with 95% O2/5% CO2). Immediately thereafter, rats were de-
capitated, their brains were removed, and hemisected along the long-
itudinal fissure, and each hemisphere was prepared for slicing by
making a series of blocking cuts. Specifically, dorsal hippocampal slices
were prepared by resting the hemisected brain on its ventral surface
and making a ∼45° blocking cut (between the sagittal and coronal
planes) at the level of the striatum, which subsequently served as the
mounting surface (for a more detailed description, please see
(Dougherty et al., 2012)). Slices from the ventral hippocampus were
prepared by resting the hemisected brain on its sagittal surface and
making a single blocking cut along the horizontal plane at the level of
the dorsal hippocampus, which subsequently served as the mounting
surface. 350 μm thick transverse slices were then prepared using a vi-
brating microtome (Vibratome 1000; McCormick Scientific, St Louis,
MO, USA) and allowed to recover in a holding saline of the following
composition (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 2
CaCl2, 2 MgCl2, 12.5 dextrose, 1.3 ascorbic acid, and 3 sodium pyruvate
(continuously bubbled with 95% O2/5% CO2), for 15 min at 35 °C, and
then at room temperature for an additional 45 min.
2.2. Electrophysiology
Somatic whole-cell current-clamp recordings were performed on
CA1 pyramidal neurons from submerged slices bathed in a flowing
(1–2 mL/min), heated (31–33 °C) artificial cerebrospinal solution
(aCSF) of the following composition (in mM): 125 NaCl, 3 KCl, 1.25
NaH2PO4, 25 NaHCO3, 2 CaCl2, 1 MgCl2, 12.5 dextrose, 1.3 ascorbate,
and 3 sodium pyruvate (continuously bubbled with 95% O2/5% CO2).
Neurons were visually identified using a Zeiss Axioskop FS microscope
equipped with infrared differential interference contrast optics coupled
with an infrared camera and television monitor (DAGE-MTI, Michigan
City, IN, USA). Electrodes (4–6 MΩ) were pulled from borosilicate ca-
pillary glass using a P-87 Flaming–Brown micropipette puller (Sutter
M.C. Evans, K.A. Dougherty
Instruments, San Fransisco, CA, USA), and filled with an internal so-
lution with the following composition (in mM): 120 potassium gluco-
nate, 20 KCl, 10 HEPES, 4 NaCl, 4 Mg-ATP, 0.3 Na-GTP, and 7 (Tris)2-
phosphocreatine (pH 7.3, adjusted with KOH; 300 mOsm, adjusted with
sucrose). Whole-cell current-clamp recordings were performed using a
Dagan BVC-700A amplifier (Dagan, Minneapolis, MN, USA) under the
control of a MacMini computer (Apple Computer Company, Cupertino,
CA, USA) through an ITC-18 computer interface (InstruTech, Port
Washington, NY, USA). Signals from were low-pass filtered at 3 kHz and
sampled at 10 kHz for subthreshold experiments, whereas signals from
suprathreshold experiments were low-pass filtered at 5 kHz and sam-
pled at 40 kHz. Neurons were allowed to stabilize for at least five
minutes after break in, and the pipette capacitance and access re-
sistance were compensated throughout the experiment prior to each
recording. Experiments were terminated if the series resistance ex-
ceeded 30 MΩ, and small DC current injections were often used to
maintain the membrane potential at −65 mV. Membrane potentials
have not been adjusted to account for a liquid junction potential of
approximately 8 mV. Neurobiotin (0.1–0.2%; Vector Laboratories,
Burlingame, CA) was added to the internal solution in order to facilitate
the subsequent visualization of individual neurons and to verify intact
apical and basal dendritic trees. After each experiment, slices were fixed
in 0.1 M phosphate buffer with 3% gluteraldehyde, stored at 4 °C for at
least 48 h, and processed using an avidin-HRP system activated by
diaminobenzidine (DAB; Vector Laboratories). Processed slices were
mounted in glycerol and visualized using a compound microscope with
a 10X objective.
2.3. Drugs and solutions
All experiments were performed in an aCSF supplemented with
20 μM 6,7-dinitroquinoxaline 2,3-dione (DNQX; Abcam, Cambridge,
MA, USA) and 2 μM Gabazine (Abcam) to block AMPA receptors and
GABAA receptors, respectively, and thereby suppress spontaneous ac-
tivity within the slice. Carbamazepine (Sigma-Aldrich, St Louis, MO,
USA) was dissolved in DMSO (Sigma-Aldrich) to yield a 100 mM stock
solution, which was subsequently aliquoted and frozen until needed.
Carbamazepine aliquots were diluted 1000-fold into aCSF to yield
100 μM carbamazepine and 0.1% DMSO. This CBZ concentration is
approximately 2–3X the plasma concentration required to suppress
hind limb extension in the maximal electroshock seizure test in rats
(Bialer et al., 2004). DMSO was diluted 1000-fold in aCSF to yield 0.1%
DMSO solutions for vehicle control experiments.
2.4. Data analysis
Suprathreshold parameters, such as the threshold voltage (Vthr),
maximal dV/dt, and action potential peak (APpeak), were determined
through phase–plane analysis, where Vthr was defined as the membrane
potential were the dV/dt first exceeded 20 mV/ms. The action potential
size was defined as the difference between the APpeak and Vthr, and the
action potential half-width was defined as the width of the action po-
tential waveform at one half of the action potential size. The spike
frequency adaptation (SFA) ratio was determined from action potential
trains elicited by one second long current injections, where the current
magnitude was varied to produce trains containing 10–12 action po-
tentials. The SFA was then calculated by dividing the duration of the
final interspike interval (ISI) by the duration of the first ISI. The input
resistance (Rin) was determined as the slope of the voltage–current plot
constructed from the steady-state voltage responses to a family of
current injections ranging from −50 to 50 pA (only the linear region of
these plots were used for determining the Rin). The membrane time
constant (τm) was determined by averaging the voltage responses to
100, 1 ms square current injections (100 pA), and fitting the average
response with a double exponential curve. The slower of the two time
constants was taken as τm.
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Epilepsy Research 145 (2018) 63–72
2.5. Statistical evaluation
Statistical significance between paired groups was determined using
a Paired Student’s t test when n ≥ 7, and a Wilcoxon Signed-Rank test
when n < 7, the data were not normally distributed, or the variances
were unequal. Statistical significance between independent groups was
determined using Student’s t test when n ≥ 7, and a Wilcoxon Ranked-
Sum test when n < 7, the data were not normally distributed, or the
variances were unequal. Differences between firing frequency versus
current injection (F–I) relationships were determined using a Two-Way
Repeated Measures ANOVA test, followed by a Bonferroni correction
for multiple comparisons. Paired F–I relationships were evaluated using
repeated measures in two dimensions (time and current injection),
whereas unpaired F–I relationships were evaluated using repeated
measures in one dimension (current injection). Spearman’s Rank
Correlation test was used to evaluate correlation between datasets.
Unless otherwise stated, statistical analyses and graphical displays were
accomplished using IGOR Pro v6.35 (Wavemetrics, Lake Oswego, OR,
USA). Two-Way ANOVA tests and correlation analyses were performed
using Prism 7 (GraphPad Software, La Jolla, CA, USA). All hypothesis
testing was conducted with α = 0.05, and the specific statistical tests
used to evaluate each dataset are indicated in the corresponding figure
legends. All data are presented as SEM, unless otherwise stated.
3. Results
3.1. Baseline parameters
In order to evaluate the influence of carbamazepine along the
longitudinal hippocampal axis, we performed whole-cell current-clamp
recordings on CA1 pyramidal neurons from DHC and VHC slices pre-
pared from adult male rats (4.7 ± 0.5 months old; 403 ± 12 g). DHC
slices were sampled from a range of locations along the longitudinal
hippocampal axis that included both the dorsal hippocampus and the
dorsal intermediate hippocampus (dIHC; as defined by Malik et al.,
2016), whereas VHC slices were taken mostly from the ventral inter-
mediate hippocampus (vIHC), rather than the ventral hippocampus
near the temporal pole (Malik et al., 2016). CA1 pyramidal neurons
from this segment of the longitudinal hippocampal axis (DHC–dIHC–-
vIHC) are generally similar with regard to their morphology and
baseline intrinsic parameters, such as Rin (at −65 mV), τm (at −65 mV),
and Vthr (at −65 mV) (Malik et al., 2016). This observation stands in
contrast to the region of the VHC closest to the temporal pole, where
CA1 pyramidal neurons exhibit a significantly different morphological
and electrophysiological phenotype (Malik et al., 2016; Dougherty
et al., 2012). Therefore, we limited our study to the DHC–dIHC–vIHC
segment of the longitudinal hippocampal axis, and will hereafter refer
to the vIHC as the VHC. Accordingly, representative DHC and VHC
slices, with corresponding representative filled CA1 pyramidal neurons
are displayed in Fig. 1A–B. Baseline parameters such as RMP, Rin (at
−65 mV), Rin (at RMP), and τm (at −65 mV) were not significantly
different between DHC and VHC neurons (DHC RMP = −63.4 ±
0.9 mV, n = 15, Rin (at −65 mV) = 58.0 ± 2.5 MΩ, n = 15, Rin (at
RMP) = 59.6 ± 3.9 MΩ, n = 11, τm (at –65 mV) = 20.0 ± 1.1 ms,
n = 10; VHC RMP = −62.0 ± 0.8 mV, n = 15, Rin (at
−65 mV) = 61.8 ± 3.4 MΩ, n = 15, Rin (at RMP) = 71.5 ± 8.9 MΩ,
n = 11, τm (at –65 mV) = 21.0 ± 1.9, n = 10; Fig. 1C–E). These values
were similar to those reported in a previous study using adult rats under
similar conditions (i.e., without blocking NMDA receptors), although
the Rin values for VHC neurons were slightly lower than previously
reported (Clemens and Johnston, 2014). This is likely due to the
longitudinal location of our VHC slices (see above). The voltage
threshold (Vthr) of single action potentials elicited by current injections
from −65 mV were also found to be indistinguishable between DHC
and VHC neurons in both the 10 ms or 50 ms timeframes (Fig. 1F; DHC
Vthr (10 ms) = −49.2 ± 1.0 mV, n = 13, Vthr (50 ms) = −47.0 ±
M.C. Evans, K.A. Dougherty Epilepsy Research 145 (2018) 63–72

Fig. 1. Baseline properties of CA1 pyramidal neurons across the longitudinal hippocampal axis of adult rats. A–B) Representative slices (left) and filled CA1
pyramidal neurons (right) from the dorsal (A) and ventral (B) hippocampus. C–E) The resting membrane potential (C), Rin (at −65 mV) (D; circles), the Rin (at RMP)
(D; squares), and the τm (at −65 mV) (E) of DHC (blue) and VHC neurons (black) were not significantly different (Student’s t-test, p > 0.05). F) The voltage threshold
measured 10 ms (circles) and 50 ms (squares) after the onset of the current stimulus were not significantly different between DHC (blue) and VHC (black) neurons
(Student’s t-test, p > 0.05). The voltage threshold for VHC neurons was significantly more depolarized in the 50 ms timeframe than in the 10 ms timeframe (Students’s
t-test, p < 0.05), whereas the voltage threshold was not significantly different for DHC neurons between these timeframes (Student’s t-test, p > 0.05). Average
values are represented as horizontal lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

0.8 mV, n = 13; VHC Vthr (10 ms) −49.6 ± 0.6 mV, n = 12, Vthr
(50 ms) = −46.5 ± 0.8 mV, n = 12). Although some subtle gradients
in intrinsic excitability are likely present, these observations demon-
strate that several key factors of intrinsic excitability are generally si-
milar along a significant portion of the longitudinal hippocamal axis.
This overall similarity serves as a foundation for investigating the in-
fluence of AEDs across this region of the longitudinal hippocampal axis.
3.2. Carbamazepine-induced suppression of repetitive firing is greater in
DHC neurons than VHC neurons
We evaluated the effect of carbamazepine on repetitive firing in
DHC and VHC neurons before, and 20–60 min after applying 100 μM
CBZ through the bath solution (solution exchange was conducted at
RMP, which was allowed to float throughout the wash-in period).
100 μM CBZ significantly reduced the firing frequency for action po-
tential trains elicited by one-second long current injections from
−65 mV (Fig. 2A–C). This effect was especially pronounced in DHC
neurons, which often ceased firing after just a few action potentials
(Fig. 2A, left column). In contrast, VHC neurons tended to fire
throughout the current injection, albeit at a lower frequency (Fig. 2A,
right column). Although the F–I baseline relationships for DHC and
VHC neurons were indistinguishable (Fig. 2D), the F–I relationship for
DHC neurons was significantly lower than their VHC counterparts in
the presence of 100 μM CBZ (Fig. 2E). This differential suppression of
the F–I relationship was not observed in vehicle control experiments,
where only 0.1% DMSO was applied to DHC and VHC neurons (Sup-
plementary Fig. 1).
We next extended the duration of the current injection from one
second to ten seconds in order to examine the influence of carbama-
zepine on firing for DHC and VHC neurons throughout prolonged action
potential trains. Accordingly, both DHC and VHC neurons fired
throughout the 10 s, 250 pA current injection in the absence of carba-
mazepine (Fig. 3A). Exposure to 100 μM CBZ significantly reduced the
firing frequency of both DHC neurons and VHC neurons, although VHC
neurons continued to fire throughout the current stimulus, whereas
DHC neurons often fired a few action potentials at the beginning of the
current stimulus before ceasing firing altogether (Fig. 3A–C). As in the
shorter, one second timeframe, 100 μM CBZ induced a significantly
greater suppression in the F–I relationships for DHC neurons than their
VHC counterparts, despite indistinguishable baseline F–I relationships
(Fig. 3D–E). There was no significant reduction of the F–I relationship
in 0.1% DMSO control experiments (Supplementary Fig. 2). Moreover,
the firing frequency in the presence of 0.1% DMSO increased over time,
yielding a fold change in firing frequency (10 s, 250 pA current injec-
tions) greater than one for both DHC and VHC neurons (Fig. 4; DHC
Fold Change (DMSO) = 1.63 ± 0.16, n = 6; VHC Fold Change
(DMSO) = 1.37 ± 0.18, n = 5). When compared to 0.1% DMSO con-
trol experiments, 100 μM CBZ significantly reduced the firing frequency
of both DHC and VHC neurons (Fig. 4; DHC Fold Change
(CBZ) = 0.13 ± 0.06, n = 8; VHC Fold Change (CBZ) = 0.65 ± 0.13,
n = 7). The fold change in firing frequency in response to 100 μM CBZ
was significantly lower for DHC neurons than their VHC counterparts
(Fig. 4).
3.3. Carbamazepine influences subthreshold and supratheshold properties
of CA1 pyramidal neurons from the dorsal hippocampus
In order to understand the mechanism(s) driving the dispropor-
tionate effect of CBZ on DHC neurons, we measured carbamazepine’s
effect on single action potentials in DHC and VHC neurons. Single ac-
tion potentials were elicited by brief (10 ms) current injections of
variable amplitude, such that the first action potential occurred within
a ± 1 ms time window centered 8 ms following the onset of the current
stimulus before and after 100 μM CBZ wash in (Fig. 5A; Table 2). This
short timeframe was chosen to minimize the influence of slowly acti-
vating and inactivating ion channels. Under these conditions, baseline
parameters from DHC and VHC neurons were indistinguishable

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M.C. Evans, K.A. Dougherty Epilepsy Research 145 (2018) 63–72

Fig. 2. Carbamazepine-induced suppression of repetitive firing in response to short current injections. A) Representative traces from DHC (left) and VHC
(right) neurons elicited by 1 s, 330 pA current injections from −65 mV before (upper traces) and after (lower traces) exposure to 100 μM carbamazepine. B–C) F–I
relationships for DHC (B) and VHC (C) neurons before (hollow circles) and after (filled circles) exposure to 100 μM carbamazepine. Carbamazepine significantly
suppressed repetitive firing for DHC neurons and VHC neurons (Two-way repeated measures ANOVA; Bonferroni test for multiple comparisons, p < 0.05). D–E) F–I
relationships of DHC and VHC neurons before (D) and after (E) exposure to 100 μM carbamazepine. In the presence of 100 μM CBZ, the F–I relationship was
significantly lower for DHC neurons than VHC neurons (Two-way repeated measures ANOVA; Bonferroni test for multiple comparisons, p < 0.05).

(Table 2), except for the maximum dV/dt, which was significantly
lower for VHC neurons than DHC neurons (DHC max dV/
dt = 437.1 ± 16.5 mV/ms, n = 13; VHC max dV/dt = 371.2 ±
12.8 mV/ms, n = 12) Subsequent exposure to 100 μM CBZ did not
significantly alter the voltage threshold (DHC ΔVthr = −1.2 ± 2.0 mV,
n = 8; VHC ΔVthr = −0.6 ± 0.7 mV, n = 6), action potential size
(DHC ΔAPsize = −3.5 ± 1.6 mV, n = 8; VHC ΔAPsize = −3.8 ±
2.0 mV, n = 6), and action potential halfwidth (DHC ΔAPhw =
0.02 ± 0.02 ms, n = 8; VHC ΔAPhw = 0.04 ± 0.03 ms, n = 6) for ei-
ther DHC or VHC neurons (Fig. 5; Table 2). However, the maximal dV/

Fig. 3. Carbamazepine-induced suppression of repetitive firing in response to long current injections. A) Representative traces from DHC (left) and VHC
(right) neurons elicited by 10 s, 250 pA current injections from −65 mV before (upper traces) and after (lower traces) exposure to 100 μM carbamazepine. B–C) F–I
relationships for DHC (B) and VHC (C) neurons before (hollow circles) and after (filled circles) exposure to 100 μM carbamazepine. Carbamazepine significantly
suppressed repetitive firing for DHC neurons and VHC neurons (Two-way repeated measures ANOVA; Bonferroni test for multiple comparisons, p < 0.05). D–E) F–I
relationships of DHC and VHC neurons before (D) and after (E) exposure to 100 μM carbamazepine. In the presence of 100 μM CBZ, the F–I relationship was
significantly lower for DHC neurons than VHC neurons (Two-way repeated measures ANOVA; Bonferroni test for multiple comparisons, P < 0.05).

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M.C. Evans, K.A. Dougherty Epilepsy Research 145 (2018) 63–72

Fig. 4. Summary of carbamazepine’s effect on firing frequency. The fold
change in firing frequency (relative to the baseline firing frequency) for action
potential trains elicited by 10 s, 250 pA current injections, is significantly lower
in the presence of 100 μM carbamazepine for both DHC and VHC neurons re-
lative to 0.1% DMSO controls (Wilcoxon’s Ranked Sum Test, p < 0.05). The
fold change in firing frequency is significantly lower for DHC neurons than VHC
neurons following 100 μM CBZ exposure (Wilcoxon’s Ranked Sum Test,
p < 0.05), whereas there was no difference in the fold change in firing fre-
quency following 0.1% DMSO exposure (Wilcoxon’s Ranked Sum Test,
p > 0.05).
dt was significantly reduced for both DHC and VHC neurons in the
presence of 100 μM CBZ (Fig. 5D; DHC Δmax dV/dt = −41.6 ±
13.0 mV/ms, n = 8; VHC Δmax dV/dt = −27.7 ± 7.2 mV/ms, n = 6).
Despite this effect, single action potentials were mostly unaffected by
the presence of 100 μM CBZ, which is consistent with carbamazepine’s
classical mechanism of action as a use-dependent sodium channel
blocker (Ragsdale et al., 1991).
Although single action potentials were mostly unaffected, carba-
mazepine had a profound effect on action potential trains for both DHC
and VHC neurons (Figs. 2 and 3). Accordingly, we examined carba-
mazepine’s influence on spike frequency adaptation, by calculating the
SFA ratio from action potential trains containing 10–12 action poten-
tials (Fig. 6A). The baseline SFA ratio was significantly larger for DHC
neurons compared to VHC neurons (Fig. 6B; DHC SFA ratio = 2.95 ±
0.31, n = 11; VHC SFA ratio = 1.39 ± 0.19, n = 10), which is con-
sistent with previous reports (Malik et al., 2016). However, in the
presence of 100 μM CBZ, it became impossible to elicit the 10–12 action
potentials required to determine the SFA ratio in all but two DHC
neurons–the SFA ratio for these two neurons increased 2.41 and 1.67
fold. Alternatively, 100 μM CBZ did not significantly alter the SFA ratio
for VHC neurons (Fig. 6B; VHC ΔSFA ratio = 1.59 ± 0.13, n = 5), and
no significant effect was observed in 0.1% DMSO control experiments
(Fig. 6B; DHC ΔSFA ratio = 0.72 ± 0.06, n = 5; VHC ΔSFA
ratio = 0.88 ± 0.24, n = 4). These observations demonstrate that
carbamazepine induces a profound spike frequency adaptation in DHC
neurons, but spares their VHC counterparts.
Carbamazepine’s effect was not limited to supratheshold properties.
Surprisingly, carbamazepine influenced the subthreshold properties of

Fig. 5. Carbamazepine does not significantly alter single action potentials. A) Single action potentials elicited by 10 ms current injections before and after
exposure to 100 μM CBZ in DHC (upper traces) and VHC (lower traces) neurons. B–C) The threshold voltage (B) and action potential size (C) were not significantly
different following 100 μM CBZ exposure for DHC (blue circles; Paired Student’s t-test, p > 0.05) or VHC (black circles; Wilcoxon’s Signed Rank test, p > 0.05)
neurons. D) Carbamazepine significantly reduced the max dV/dt for both DHC (Paired Student’s t-test, p < 0.05) and VHC neurons (Wilcoxon’s Signed Rank test,
p < 0.05). E) The action potential halfwidth was not significantly different following carbamazepine exposure for either DHC (Paired Student’s t-test, p > 0.05) or
VHC neurons (Wilcoxon’s Signed Rank test, p > 0.05). Average values are represented as horizontal lines. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)

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M.C. Evans, K.A. Dougherty Epilepsy Research 145 (2018) 63–72

Table 2
Single action potential parameters.
Voltage Threshold (mV) Maximum dV/dt (mV/ms) AP size (mV) AP halfwidth (ms)

DHC CBZ baseline −49.3 ± 1.3 (8) 438.9 ± ac

18.1 (8) 90.9 ± 1.4 (8) 0.87 ± 0.04 (8)
DHC CBZ wash-in −50.5 ± 1.8 (8) 397.2 ± 15.3a (8) 87.4 ± 1.6 (8) 0.88 ± 0.05 (8)
DHC DMSO baseline −49.0 ± 1.7 (5) 434.3 ± 34.8 (5) 86.1 ± 4.6 (5) 0.88 ± 0.03 (5)
DHC DMSO wash-in −52.3 ± 0.8 (5) 398.5 ± 21.2 (5) 87.3 ± 2.0 (5) 0.91 ± 0.04 (5)
VHC CBZ baseline −50.3 ± 0.6 (6) 370.2 ± 17.2bc (6) 88.7 ± 1.45 (6) 0.93 ± 0.04 (6)
VHC CBZ wash-in −50.9 ± 0.4 (6) 342.6 ± 18.9b (6) 84.9 ± 2.6 (6) 0.97 ± 0.05 (6)
VHC DMSO baseline −48.4 ± 1.1 (5) 383.5 ± 20.9 (5) 86.1 ± 2.44 (5) 0.87 ± 0.02 (5)
VHC DMSO wash-in −50.4 ± 1.3 (5) 374.7 ± 17.3 (5) 80.1 ± 2.7 (5) 0.79 ± 0.06 (5)

The parameters of single action potentials elicited approximately 8 ms after the onset of a square pulse from −65 mV are displayed before (baseline), and after
exposure to either 100 μM CBZ or 0.1% DMSO. Statistical significance was determined using either a paired Student’s t test (n ≥ 7) or a Wilcoxon’s Signed Rank test
(n < 7), with p < 0.05. Significant differences are indicated by superscripted letters, and n values are indicated in parentheses.

Fig. 6. Carbamazepine-induced enhancement of spike frequency adaptation across the longitudinal hippocampal axis. A) The SFA ratio was determined by
dividing the duration of the final ISI (dotted lines) by the first ISI (solid lines) for one second long action potential trains containing 10–12 action potentials. B) The
baseline SFA ratio was significantly higher for DHC neurons (blue symbols) than VHC neurons (black symbols) for both 0.1% DMSO control (squares; Wilcoxon’s
Ranked Sum test, p < 0.05) and 100 μM CBZ experiments (circles; Wilcoxon’s Ranked Sum test, p < 0.05). Exposure to 100 μM CBZ induced a profound spike
frequency adaptation in DHC neurons that made it impossible to elicit 10–12 action potentials in the one second time frame in all but two neurons, whereas VHC
neurons continued to fire throughout the one second interval, although 100 μM CBZ did not significantly alter the SFA ratio (Wilcoxon’s Signed Rank test, p > 0.05).
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

DHC neurons, as 100 μM CBZ induced a small, but significant depo-
larization of RMP for DHC neurons, but not VHC neurons (Fig. 7A; DHC
ΔRMP = 3.0 ± 1.1 mV, n = 9; VHC ΔRMP = −0.2 ± 0.6 mV, n = 8).
However, the presence of 100 μM CBZ did not significantly alter the Rin
(at − 65 mV) (Fig. 7B–C; DHC ΔRin (at -65 mV) = -0.8 ± 5.6 MΩ,
n = 6; VHC ΔRin (at −65 mV) = 10.1 ± 9.7 MΩ, n = 7), and no
changes in either the RMP nor the Rin (at −65 mV) were observed
during 0.1% DMSO control experiments (DHC ΔRMP = 1.3 ± 0.8 mV,
ΔRin (at -65 mV) = 7.6 ± 7.2 MΩ, n = 6; VHC ΔRMP = −1.3 ±
0.8 mV, ΔRin (at −65 mV) = 5.1 ± 8.6 MΩ, n = 7). Furthermore,
100 μM CBZ did not alter τm (at −65 mV) for DHC neurons (Fig. 7D–E;
Δτm (at −65 mV) = 0.8 ± 3.3 ms, n = 6), but induced a small, but
significant increase in the τm (at −65 mV) for VHC neurons (Fig. 7D–E;
VHC Δτm (at −65 mV) = 3.1 ± 2.0 ms, n = 6). No significant differ-
ences were observed for τm (at −65 mV) following exposure to 0.1%
DMSO (DHC Δτm (at −65 mV) = 5.9 ± 2.1, n = 4; VHC Δτm (at
−65 mV) = 2.1 ± 0.8 ms, n = 4). Interestingly, cells that depolarized
in the presence of 100 μM CBZ tended to show a greater suppression of
their F–I relationships, as the ΔRMP was significantly correlated with
the fold change in firing frequency along the longitudinal hippocampal
axis (Fig. 8A). Neither the suprathreshold nor the subthreshld effect of
carbamazepine significantly correlated with the original RMP, at which
100 μM CBZ was applied to the slice (data not shown). Together, these
observations demonstrate that carbamazepine exerts a unique influence
over the intrinsic excitability of CA1 pyramidal neurons from the DHC.
4. Discussion
4.1. Summary
In this study, we investigated the influence of carbamazepine on CA1
pyramidal neurons across the longitudinal hippocampal axis, and have
found that carbamazepine 1) significantly suppresses repetitive firing in
DHC neurons through an enhancement of spike frequency adaptation
(Figs. 2–4 and 6), 2) induces a slight depolarization of the RMP in DHC
neurons (Fig. 7), and 3), that these two effects were significantly corre-
lated across the longitudinal hippocampal axis (Fig. 8). Carbamazepine
also suppressed repetitive firing in VHC neurons, but this effect was
significantly smaller than their DHC counterparts (Figs. 2–4). Moreover,
no change in the RMP was observed following 100 μM CBZ exposure for
VHC neurons (Fig. 7A). Together, these observations suggest a unique
sensitivity to carbamazepine for DHC neurons relative to their VHC
counterparts. The potential mechanisms and physiological implications
of these findings are further discussed below.
4.2. Comparison with previous work
Interestingly, this regional difference in carbamazepine sensitivity
emerged from a population of CA1 pyramidal neurons with similar
baseline intrinsic properties (e.g., RMP, Rin (at −65 mV), Rin (at RMP),
τm (at −65 mV), and Vthr; Fig. 1). At first glance, this baseline similarity
between DHC and VHC neurons seemingly contradicts the significant
differences in intrinsic excitability reported across the longitudinal

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M.C. Evans, K.A. Dougherty Epilepsy Research 145 (2018) 63–72

Fig. 7. 100 μM carbamazepine influences subthreshold properties of CA1 pyramidal neurons. A) The RMP of DHC neurons (blue circles), but not VHC neurons
(black circles) was significantly more depolarized following exposure to 100 μM CBZ (Paired Student’s t-test, p < 0.05). B–C) the Rin (at −65 mV) was not sig-
nificantly different following exposure to 100 μM CBZ for either DHC (B blue circles; C blue traces), or VHC neurons (B black circles; C black traces; Paired Student’s t-
test, p > 0.05). D–E) the τm (at −65 mV) was not significantly different following exposure to 100 μM CBZ for DHC neurons (D blue circles; E left, blue/gray traces;
Wilcoxon’s Signed Rank test, p > 0.05) but showed a small, yet significant increase for VHC neurons (D black circles; E right, black/gray traces; Wilcoxon’s Sign Rank
test, p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8. Correlation between carbamazepine’s subthreshold and supra-
threshold effects across the longitudinal hippocampal axis. DHC (blue
circles) and VHC neurons (black circles) were combined in order to evaluate the
correlation between the ΔRMP with the fold change in firing frequency for CA1
pyramidal neurons across the longitudinal hippocampal axis. This relationship
was described assuming a linear function with the following parameters: y-
intercept = 3.2 mV, slope = −4.1 mV/fold change, and correlation was eval-
uated using Spearman’s Rank Correlation test (R = −0.54, p = < 0.05, n = 15).
(For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
hippocampal axis (Dougherty et al., 2012; Hönigsperger et al., 2015;
Malik et al., 2016; Kim and Johnston, 2015; Milior et al., 2016).
However, our current experimental conditions differ from those re-
ported in previous studies in several important ways: 1) The rats used
for this study were significantly older than those used in previous stu-
dies (Dougherty et al., 2012, 2013; Malik et al., 2016; Kim and
Johnston, 2015), 2) NMDA receptor blockers were omitted from our
cocktail of synaptic blockers, and 3) slices for this study were prepared
using a slightly different blocking strategy than previous studies, which
doesn’t sample the extreme ventral hippocampus near the temporal
pole. Recent work by Clemens and Johnston (2014), using adult rats
and a similar synaptic blocker cocktail (i.e., without NMDA blockers)
reported an ∼3 mV difference between the average RMPs, and an ∼
20 MΩ difference in the Rin (at −65 mV) of DHC and VHC neurons
(Clemens and Johnston, 2014). Our baseline RMP and Rin (at −65 mV)
values matched those previously reported by Clemens and Johnston
(2014) for DHC neurons, whereas our Rin (at −65 mV) was lower than
previously reported for VHC neurons (62 MΩ versus ∼80 MΩ from
Clemens and Johnston (2014)). This discrepancy likely stems from the
relative longitudinal position of the slices used for this study. Accord-
ingly, Malik et al. (2016) have recently reported tends in intrinsic ex-
citability along the longitudinal axis of young adult rats, wherein the
Rin (at −65 mV) remains constant around 60 MΩ along the dorsal
portion of the longitudinal axis until jumping to about 80 MΩ near the
temporal pole (Malik et al., 2016). The slices used in this study were
prepared mostly from the vIHC, rather than the extreme VHC, which
places our observations in good agreement with those previously re-
ported by Malik et al. (2016). This reasoning also accounts for the si-
milarity in Vthr that we observed across the longitudinal hippocampal
axis, as Vthr remains similar across the DHC, dIHC, and vIHC, before
depolarizing in the extreme VHC (Malik et al., 2016). The similarity in
RIn (at −65 mV) and Vthr along this region of the longitudinal hippo-
campal axis spares our interpretation from the potentially confounding
influence of differences in baseline intrinsic excitability.

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M.C. Evans, K.A. Dougherty
4.3. Potential mechanisms
The main finding of this work is the region-specific suppression of
repetitive firing of CA1 pyramidal neurons along the longitudinal hip-
pocampal axis in response to 100 μM CBZ. In the presence of carba-
mazepine, the F–I relationships for DHC neurons were significantly
more suppressed than their VHC counterparts in both the one and 10 s
time frames (Figs. 2 and 3), despite having similar baseline intrinsic
properties (see above). Moreover, single action potentials were gen-
erally unaffected by the presence of 100 μM CBZ, although there was a
slight reduction in the maximal dV/dt for both DHC and VHC neurons
(Fig. 5). Instead, carbamazepine influenced action potential trains by
augmenting spike frequency adaptation in DHC neurons (Figs. 2, 3 and
6). This enhancement of spike frequency adaptation was so profound,
that most DHC neurons fired only 1–5 action potentials shortly after the
stimulus onset, which precluded the determination of the SFA ratio for
these neurons. This difference between carbamazepine’s effect on DHC
and VHC neurons is striking, and raises questions about mechanistic
differences between these two populations of neurons. Recently, Malik
et al. (2016) reported a gradient in spike frequency adaptation along
the longitudinal hippocampal axis, with DHC neurons showing the
greatest degree of adaptation (Malik et al., 2016). Our work corrobo-
rates this baseline difference in spike frequency adaptation, and sug-
gests that carbamazepine exaggerates this pre-existing difference in
spike frequency adaptation across the longitudinal hippocampal axis,
thereby implying some difference in the molecular targets present in
these neurons.
Carbamazepine’s classical mechanism of action involves the use-
dependent blockade of voltage-gated sodium channels. This progres-
sively limits the fraction of available sodium channels throughout an
action potential train, thereby depolarizing threshold and prolonging
the interspike interval, which results in spike frequency adaptation.
This effect relies primarily on the loss of the transient component of
sodium currents mediated by voltage-gated sodium channels, due to a
hyperpolarizing shift in the steady-state inactivation curve and corre-
spondingly slowed recovery from inactivation. However, Uebachs et al.
(2010) have reported a paradoxical carbamazepine-induced effect on
the persistent sodium current that relies on sodium channel subunit
composition (Uebachs et al., 2010). Specifically, carbamazepine in-
duces a hyperpolarizing shift in the conductance vs voltage relationship
(G–V curve) of the persistent sodium current in CA1 pyramidal neurons
from mice lacking the β1 auxiliary subunit. This augmentation of the
subthreshold sodium current accelerates the depolarizing envelope
between action potentials, thereby decreasing the interspike interval
and counteracting the suppression of repetitive firing in these neurons.
The resulting F–I relationships for Scn1b knock-out mice are remarkably
unchanged by exposure to 100 μM CBZ (Uebachs et al., 2010). Our
results are generally consistent with such a mechanism, as the sig-
nificantly reduced carbamazepine sensitivity of VHC neurons mirrored
the effect of 100 μM CBZ on CA1 neurons from Scn1b knock-out mice,
whereas carbamazepine’s suppression of repetitive firing in DHC neu-
rons resembled the Scn1b wild type mouse (Uebachs et al., 2010). Im-
portantly, this potential mechanism is undergirded by the observation
that β1, β3, and β4 sodium channel subunits were expressed more
abundantly in the DHC relative to the VHC (Table 1), with no difference
in β2-subunit expression or any of the relevant sodium channel α-sub-
unit genes (i.e., Scn1A/Nav1.1, Scn2a1/Nav1.2, Scn3 A/Nav1.3, and
Scn8 A/Nav1.6; Table 1). This observation implies a gradient in β1-
subunit expression along the longitudinal hippocampal axis, which
could modify carbamazepine’s efficacy along this axis, in a manner
consistent with our results. The potential influence of the β3 and β4-
subunits on carbamazepine efficacy in the DHC, however, remains
unexplored.
In addition to the suppression of the F–I relationship, we also ob-
served a slight depolarization of the RMP in DHC neurons, which cor-
related with the decreased firing frequency across all cells (DHC and
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Epilepsy Research 145 (2018) 63–72
VHC; Figs. 7 and 8). Interestingly, the depolarization of RMP occurred
in DHC neurons, rather than in VHC neurons, where a depolarization of
RMP might be expected if carbamazepine was augmenting the persis-
tent sodium current. This suggests that the effect on RMP is either
mechanistically unrelated to the decreased firing frequency in DHC
neurons, or that our inferred mechanism involving the persistent so-
dium current is incorrect, and that carbamazepine’s dual effects on DHC
neurons are coupled through an unknown mechanism. We favor the
former explanation, as DHC neurons are known to express secondary
targets for carbamazepine: A1 adenosine receptors (Lee et al., 1983;
Kim and Johnston, 2015). Carbamazepine interacts with A1 adenosine
receptors, which, through their coupling with GIRK channels, hy-
perpolarize the membrane potential of DHC neurons (Weir et al., 1984;
Kim and Johnston, 2015). The slight depolarization of RMP observed in
DHC neurons could plausibly stem from carbamazepine’s interaction
with this system, although additional work will be required to fully
explore this hypothesis.
4.4. Physiological implications
Although carbamazepine is most commonly used for seizure control
in epileptic patients, it is often used to treat non-epileptic conditions,
such as neuropathic pain (trigeminal neuralgia and diabetic neuro-
pathy), bipolar disorder, and post-traumatic stress disorder (PTSD),
among others (Spina and Perugi, 2004). Because this study was per-
formed using naive, non-epileptic tissue, the physiological ramifications
of this work should be considered in the context of carbamazepine’s
uses in non-epileptic patients. Carbamazepine is generally associated
with several negative side effects, including significant changes in
cognitive function, such as reductions in information processing speed,
attention, verbal fluency, and arithmetic (Eddy et al., 2011). Carba-
mazepine’s effect on memory is less clear, as some reports claim, im-
pairment, no effect, or even an enhancement in memory tasks (Eddy
et al., 2011). In adult rats similar to those used in this study, carba-
mazepine has been shown to impair performance in the Morris Water
Maze–a spatial learning and memory task known to rely on the DHC
(Churchill et al., 2003). This observation is generally consistent with
our main finding that carbamazepine’s effects are concentrated within
the DHC, and suggests that carbamazepine’s influence over the DHC
could impact behavior by impairing spatial learning in adult rats.
Finally, it is surprising that the carbamazepine-induced suppression
of repetitive firing is greater in the DHC, rather than in the VHC, where
seizures tend to originate, and where CA1 neurons show greater in-
trinsic excitability. This observation generally resembles another recent
study by Hönigsperger et al. (2015), which demonstrated a DHC-spe-
cific effect of retigabine–another anti-epileptic drug with a mechanism
unrelated to carbamazepine (i.e., enhancement of IM). Together, these
observations suggest that the suppression of activity in the dorsal hip-
pocampus could play a larger role in limiting seizure generation than
previously thought. However, additional experiments exploring the
effect of anti-epileptic drugs in the dorsal hippocampus of epileptic
tissue will be required to fully explore this issue.
4.5. Conclusions
In summary, we have demonstrated that acute exposure to the use-
dependent sodium channel blocker carbamazepine induces greater
suppression of repetitive firing in CA1 neurons from the DHC than the
VHC. Although single action potentials are generally unaffected, car-
bamazepine induces profound spike frequency adaptation in DHC
neurons. Moreover, carbamazepine induces a small, but significant
depolarization of the RMP for DHC neurons, but not VHC neurons, and
the magnitude of this depolarization correlates with the suppression of
repetitive firing across all regions of the longitudinal hippocampal axis
under investigation. Together, these observations provide novel insight
into carbamazepine’s side effect profile in non-epileptic tissue, and
M.C. Evans, K.A. Dougherty
could inform future work on carbamazepine’s (or related anti-epileptic
drugs) ability to suppress seizure generation in epileptic hippocampal
tissue.
Funding
This research was supported through funding from Rhodes College.
Conflicts of interest
None.
Acknowledgements
We would like to thank Dr Darrin Brager and members of the
Dougherty lab for their continuous support throughout this project.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.eplepsyres.2018.05.
014.
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