Download Regular-spiking cells in the presubiculum are hyperexcitable in a rat

J Neurophysiol 112: 2888 –2900, 2014.
First published September 10, 2014; doi:10.1152/jn.00406.2014.
Regular-spiking cells in the presubiculum are hyperexcitable in a rat model
of temporal lobe epilepsy
Saad Abbasi and Sanjay S. Kumar
Department of Biomedical Sciences, College of Medicine and Program in Neuroscience, Florida State University,
Tallahassee, Florida
Submitted 29 May 2014; accepted in final form 5 September 2014
TLE; presubiculum; cell classification; hyperexcitability; electrophysiology
THE PRESUBICULUM (PrS), DEFINED
by Brodmann in the primate
brain as area 27, is located between the subiculum and the
parasubiculum, and is known to play a critical role in spatial
information processing and memory consolidation (Boccara et
al. 2010). Additionally, research from animal models of temporal lobe epilepsy (TLE) has implicated the PrS in driving
epileptiform activity in neighboring brain structures including
the entorhinal cortex (Tolner et al. 2005). Despite its potential
to drive parahippocampal circuits, information on the physiological state of its neurons, especially under epileptic conditions, remains greatly limited.
Address for reprint requests and other correspondence: S. S. Kumar, Dept.
of Biomedical Sciences, College of Medicine, Suite 3300-B, Florida State
Univ., 1115 West Call St., Tallahassee, FL 32306-4300 (e-mail: sanjay.kumar
@med.fsu.edu).
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PrS is cytoarchietectonically distinguishable by a densely
packed pyramidal cell layer (L) II that combines with LIII to
form the superficial layers, which are separated from the
deeper layers (V-VI) by a cell sparse LIV. Input to PrS arises
from retrosplenial, cingulate, and visual cortices (Jones and
Witter 2007; Kononenko and Witter 2012; van Groen and
Wyss 1990a,b; Vogt and Miller 1983), anteroventral and laterodorsal thalamic nuclei (van Groen and Wyss 1990a), and
anterior thalamus (Shipley and Sorensen 1975; Yoder et al.
2011). PrS is reciprocally connected with the subiculum (Funahashi and Stewart 1997; Harris et al. 2001; O’Mara et al.
2001) and sends outputs to retrosplenial cortex (Van Groen and
Wyss 2003), anteroventral, anterodorsal, and laterodorsal thalamic nuclei (Seki and Zyo 1984; van Groen and Wyss 1990a;
Yoder et al. 2011), all fields of hippocampus including the
molecular layer of dentate gyrus (Kohler 1985), as well as a
prominent bilateral projection to medial entorhinal area (MEA)
(Caballero-Bleda and Witter 1994; Honda and Ishizuka 2004).
Projections from PrS to MEA terminate in LIII and LI, providing both glutamatergic and ␥-aminobutyric acid (GABA)ergic input (van Haeften et al. 1997), originating predominantly from LIII of PrS (Honda et al. 2011).
Projections from PrS to LIII of MEA are of significance to
TLE because pyramidal cells in this layer are selectively lost in
patients and rodent models of TLE (Du et al. 1993, 1995).
Additionally, unilateral ablation of PrS and the adjoining
parasubiculum prevents cell loss in MEA, providing a partial
neuroprotective effect (Eid et al. 2001). Furthermore, animal
model studies have demonstrated that stellate cells in LII of
MEA are hyperexcitable in TLE (Bear et al. 1996; Kobayashi
et al. 2003; Kumar and Buckmaster 2006; Scharfman et al.
1998) due in part to reduced inhibition mediated by loss of
local GABAergic interneurons in LIII (Kumar and Buckmaster
2006). Yet, despite the loss of primary postsynaptic targets in
MEA (LIII pyramids), PrS input to MEA is preserved and
capable of evoking epileptiform activity specific to this region
in epileptic animals (Tolner et al. 2005, 2007).
Recently, we characterized PrS neurons into distinct groups
using hierarchical cluster analysis based on electrophysiological criteria, establishing baselines for intrinsic properties mediating action potential waveform and synaptic drive in each of
the neuron types (Abbasi and Kumar 2013). However, a direct
assessment of the physiological state of PrS neurons in animals
with TLE has not been previously undertaken and the current
study addresses which of these neuronal populations are affected along with the nature of their physiological alterations.
Using the well-established pilocarpine model of TLE (Buckmaster 2004), we show that only a subset of neurons from
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Abbasi S, Kumar SS. Regular-spiking cells in the presubiculum
are hyperexcitable in a rat model of temporal lobe epilepsy. J
Neurophysiol 112: 2888 –2900, 2014. First published September 10,
2014; doi:10.1152/jn.00406.2014.—Temporal lobe epilepsy (TLE) is
the most common form of adult epilepsy, characterized by recurrent
seizures originating in the temporal lobes. Here, we examine TLErelated changes in the presubiculum (PrS), a less-studied parahippocampal structure that both receives inputs from and projects to
regions affected by TLE. We assessed the state of PrS neurons in TLE
electrophysiologically to determine which of the previously identified
cell types were rendered hyperexcitable in epileptic rats and whether
their intrinsic and/or synaptic properties were altered. Cell types were
characterized based on action potential discharge profiles followed by
unsupervised hierarchical clustering. PrS neurons in epileptic animals
could be divided into three major groups comprising of regularspiking (RS), irregular-spiking (IR), and fast-adapting (FA) cells. RS
cells, the predominant cell type encountered in PrS, were the only
cells that were hyperexcitable in TLE. These neurons were previously
identified as sending long-range axonal projections to neighboring
structures including medial entorhinal area (MEA), and alterations in
intrinsic properties increased their propensity for sustained firing of
action potentials. Frequency and amplitude of both spontaneous excitatory and inhibitory synaptic events were reduced. Further analysis
of nonaction potential-dependent miniature currents (in tetrodotoxin)
indicated that reduction in excitatory drive to these neurons was
mediated by decreased activity of excitatory neurons that synapse
with RS cells concomitant with reduced activity of inhibitory neurons.
Alterations in physiological properties of PrS neurons and their
ensuing hyperexcitability could entrain parahippocampal structures
downstream of PrS, including the MEA, contributing to temporal lobe
epileptogenesis.
EXCITABILITY OF PRESUBICULAR NEURONS IN TLE
LII-III of PrS are affected, thereby providing specific insights
into the excitability of the PrS and its potential to drive aberrant
activity in target structures.
MATERIALS AND METHODS
Animals
Slice Preparation and Electrophysiology
Rats were deeply anesthetized with urethane (1.5 g/kg ip) before
being decapitated. Horizontal slices (350 ␮m), at the level of the
hippocampus between ⫺6.6 and ⫺5.3 mm from Bregma (Paxinos and
Watson 2007), were prepared using a microslicer (VT1000S; Leica) in
a chilled (4°C) low-Ca2⫹, low-Na⫹ “cutting solution” containing the
following (in mM): 230 sucrose, 10 D-glucose, 26 NaHCO3, 2.5 KCl,
1.25 NaH2PO4, 10 MgSO4, and 0.5 CaCl2 equilibrated with a
95%-5% mixture of O2 and CO2. Slices were allowed to equilibrate in
oxygenated artificial cerebrospinal fluid (aCSF) containing the following (in mM): 126 NaCl, 26 NaHCO3, 3 KCl, 1.25 NaH2PO4, 2
MgSO4, 2 CaCl2, 0.25 L-glutamine, and 10 D-glucose (pH 7.4), first at
32°C for 1 h and subsequently at room temperature before being
transferred to the recording chamber.
Recordings were obtained at 32 ⫾ 1°C from neurons in the PrS
(average of 5.4 ⫾ 0.2 and 6.5 ⫾ 0.3 neurons per control and epileptic
rats, respectively) under Nomarski optics (Zeiss) using a visualized
infrared setup (Hamamatsu) that enabled the identification of their
location within the various lamina. Patch electrodes were pulled from
borosilicate glass (1.5-mm outer diameter, 0.75-mm inner diameter,
3– 6 M⍀) and contained the following (in mM): 105 potassium
gluconate, 30 KCl, 10 HEPES, 10 phosphocreatine, 4 MgATP, 0.3
GTP, and 20 biocytin. Internal solution pH was adjusted to 7.3 with
KOH and had an osmolarity of 300 mosM (uncorrected liquid junction potential, 12.6 mV at 32°C). The conditions applicable to recording from control animals were the same as those in epileptic animals.
Slices were maintained in oxygenated (95% O2-5% CO2) aCSF, and
drugs and chemicals were applied via the perfusate (2 ml/min).
Tetrodotoxin (TTX; Sigma, St. Louis, MO) was bath applied as
required for specific protocols.
Bipolar electrodes (CE-2C75; Fredrick Haer, Brunswick, ME) with
25-␮m tip diameters were positioned in LII or LIII of PrS in the
vicinity of the recorded neurons, and depolarizing constant current
pulses, 20- to 200-␮A in amplitude, were applied at low frequencies
(0.1– 0.3 Hz) to stimulate local afferent fibers that synapse with the
recorded neurons to evoke action potentials. The latency to response
at threshold in a subset of control and epileptic neurons was 1.9 ⫾ 0.1
(n ⫽ 14) and 2 ⫾ 0.1 ms (n ⫽ 21), respectively, typical of synaptic
delays observed during intracortical stimulation. Postsynaptic currents
and potentials were recorded using a MultiClamp 700B amplifier and
pCLAMP software (Molecular Devices, Union City, CA), filtered at
1–2 kHz (10 kHz for current clamp), digitized at 10 –20 kHz, and
stored electronically. Series resistance was monitored continuously,
and those cells in which this parameter exceeded 20 M⍀ or changed
by ⬎20% were rejected. Series resistance compensation was not used.
Whole cell current-clamp recordings were obtained in response to
injection of 1) 100 pA of depolarizing current (1–10 s in duration),
and 2) hyper- and depolarizing current steps, 600 ms in duration and
50 pA in amplitude. Spontaneous (s) and miniature (m) excitatory
postsynaptic currents (EPSCs) were obtained by holding the cell at
⫺70 mV, while inhibitory postsynaptic currents (IPSCs) were recorded at a holding potential of 0 mV, close to the reversal potential
for glutamate. The postsynaptic current data, obtained from 2-minlong continuous recordings, were analyzed with Mini Analysis (Synaptosoft, Decatur, GA). The threshold for event detection was set at
3⫻ root mean square noise level, and software-detected events
were verified visually before measuring their frequency and amplitude. Miniature postsynaptic currents (mPSCs) were isolated
with TTX (1 ␮M).
Electrophysiological and Cluster Analysis
We previously classified neurons from layers II and III of PrS using
unsupervised hierarchical cluster analysis based on electrophysiological parameters gathered from neurons in slices from control animals.
Clustering enabled establishment of a schema for neuronal classification, ultimately resulting in characterization of seven electrophysiologically distinct cell types in LII-III of PrS in control animals
(Abbasi and Kumar 2013). A portion of the control data reported in
this study is from our initial sampling of neurons and is included for
comparative purposes. Electrophysiological data for our epileptic
group were acquired from 52 neurons in acute brain slices from
animals that were confirmed epileptic. Clustering was carried out in
Origin (v. 8.6; OriginLab, Northampton, MA) with Ward’s method
and squared Euclidean distances as the distance measure (Ward 1963).
Briefly, neurons were sorted in a multidimensional space based on
similarities of the electrophysiological variables considered. Clustering began with individual neurons in separate clusters, with each
subsequent stage combining/reducing cluster number by one, based on
the distance measure between neurons, until the final stage when a
single cluster contained all of the neurons. Ward’s method uses an
ANOVA approach in determining between-cluster distances. All variables were standardized before clustering. Cluster membership was
determined by calculating the total sum of squared deviations from the
cluster mean, and clusters were combined with the intent of minimizing increases in error sum of squares (Burns 2008). The final number
of cluster groups in our analysis was determined with the Thorndike
procedure (Thorndike 1953), in which large jumps in within-cluster
distances associated with the clustering stages served to indicate
salient differences between groups and cluster number.
Cells from control and epileptic groups were analyzed identically.
Nine distinct electrophysiological parameters gathered from currentclamp recordings of single neurons were considered in the analysis.
These included the following: 1) early spike frequency adaptation, 2)
late spike frequency adaptation, 3) delay, 4) accommodative hump, 5)
sag ratio, 6) maximum firing frequency, 7) instantaneous firing frequency, 8) steady-state firing frequency, and 9) standard deviation of
the steady-state firing frequency. The choice of parameters was based
on empirical observations of the action potential waveforms of a large
population of neurons in the PrS in control animals along with
consideration of additional variables deemed most valuable in predicting group membership in alternate studies of the electrophysio-
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Sprague-Dawley rats (male: n ⫽ 10 pilocarpine treated; n ⫽ 34
controls) from postnatal (P) days 40 – 85 were used in this study. All
experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and
were approved by the Florida State University Institutional Animal
Care and Use Committee. Rats were made epileptic according to
previously described protocols for bringing up the pilocarpine model
of TLE (Buckmaster et al. 2004). Briefly, rats were treated with
pilocarpine (P38; 153 ⫾ 4 g; 380 mg/kg ip) 20 min after atropine
methylbromide (5 mg/kg ip). Diazepam (10 mg/kg ip) was administered 2 h after the onset of status epilepticus and repeated as needed.
Following recovery from status epilepticus, rats were video monitored
(40 h/wk) for spontaneous seizures. Animals used for electrophysiological experiments were confirmed epileptic (9 out of 10, of which 8
were used for electrophysiological experiments), displaying frank
spontaneous recurrent seizures scored 3 or greater on the Racine scale
(Racine 1972) on two or more observations during the 40 h/wk video
monitoring. Recordings from epileptic animals were made on average
25 days poststatus epilepticus (range: 15–35 days; 298 ⫾ 22 g), with
initial seizures observed between 9 and 25 days poststatus. Control
groups consisted of age-matched naïve rats.
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EXCITABILITY OF PRESUBICULAR NEURONS IN TLE
NeuN-biocytin Immunohistochemistry
Neurons were filled with biocytin during recording (20 mM,
included in the internal solution). To visualize biocytin-labeled neurons after recording, slices were fixed in 4% paraformaldehyde in 0.1
M phosphate buffer (PB, pH 7.4) at 4°C for at least 24 h. After
fixation, slices were stored in 30% ethylene glycol and 25% glycerol
in 50 mM PB at ⫺20°C before being processed with a whole-mount
protocol with counterstaining by NeuN immunocytochemistry. Slices
were rinsed in 0.5% Triton X-100 and 0.1 M glycine in 0.1 M PB and
then placed in a blocking solution containing 0.5% Triton X-100, 2%
goat serum (Vector Laboratories, Burlingame, CA), and 2% bovine
serum albumin in 0.1 M PB for 4 h. Slices were incubated in mouse
anti-NeuN serum (1:1,000; MAB377; Chemicon, Temecula, CA) in
blocking solution overnight. After a rinsing step, slices were incubated with the fluorophores Alexa 594 streptavidin (5 ␮g/ml) and
Alexa 488 goat anti-mouse (10 ␮g/ml; Molecular Probes, Eugene,
OR) in blocking solution overnight. Slices were rinsed, mounted on
slides, and coverslipped with Vectashield (Vector Laboratories) before being examined with a confocal microscope (TCS SP2 SE;
Leica). Brightness, contrast, and sharpness of photomicrographs were
adjusted to highlight anatomical features in Photoshop (Adobe).
Biocytin-labeled cells were visualized through a light microscope
before reconstruction of soma and dendritic arbor in Neuroleucida
(Micro-Brightfield, Colchester, VT).
All statistical values are presented as means ⫾ SE. Statistical
differences were measured with the unpaired Student’s t-test, unless
otherwise indicated.
RESULTS
We recorded from a total of 52 neurons in layers II and III
of PrS (Fig. 1, A and B) in brain slices from epileptic rats.
These, combined with 185 neurons from control animals,
enabled us to compare and assess the state of PrS in TLE. A
portion of the control data is from our previous electrophysiological and morphological characterization of cell types in
LII-LIII of the PrS (Abbasi and Kumar 2013). Interlaminar
classification within PrS (Caballero-Bleda and Witter 1993)
was as follows: PrS was identified in slices based on LII, a
cytoarchietectonically distinguishing landmark consisting of a
densely packed layer of pyramidal cells, juxtaposed with a less
densely packed LIII that extended up to a cell-sparse LIV, a
continuum of lamina dissecans in the MEA (Fig. 1B, left).
Recorded neurons were selected randomly under differential
interference contrast (DIC) optics, and laminar location was
determined visually during recording and confirmed through
biocytin-labeling and counterstaining for NeuN immunoreactivity. These sections were also used to confirm LIII cell loss in
the MEA-a histopathological feature of TLE (Fig. 1B, right).
Whole cell current-clamp recordings were used to determine
resting membrane potential of neurons (Vm), single action
potential properties, and attributes of action potential discharge
profiles in response to depolarizing and hyperpolarizing current
injections (see MATERIALS AND METHODS; Fig. 1C and Table 1).
These parameters enabled classification of neurons into distinct
groups via unsupervised hierarchical cluster analysis (Fig. 1, D
and E) and assessment of alterations in intrinsic properties of
neurons between the control and epileptic groups (Table 1).
Additionally, we measured excitability in a subset of currentclamped neurons by determining the number of action potentials evoked as a function of stimulus intensity (Fig. 2).
Stimulus intensity was measured as multiples of threshold (T),
defined as the current required for evoking a single action
potential on ⬃50% of the trails (Kumar and Buckmaster 2006).
Whole cell voltage-clamp recordings of sEPSCs and sIPSCs
from the same neurons allowed us to evaluate changes in the
excitatory and inhibitory synaptic drive to individual neurons
in control and epileptic animals (Figs. 3, 4, and 5 and Table 2).
To determine if changes in the synaptic drive to neurons were
related to alterations in number of synapses and/or probability
of neurotransmitter release, mEPSCs and mIPSCs were recorded in the presence of TTX (1 ␮M, added to aCSF bath
solution) in a subset of voltage-clamped neurons (Fig. 6 and
Table 2).
Cell-Type Classification: Cluster Analysis
We used unsupervised hierarchical cluster analysis to classify neurons based on electrophysiological parameters determined from whole cell current-clamp data. We previously
addressed the electrophysiological heterogeneity of LII-III PrS
neurons in control animals based predominantly on their action
potential discharge profiles (Abbasi and Kumar 2013). We
observed that electrophysiological properties varied significantly among the recorded neurons prompting a systematic
quantitative approach to their characterization. Cluster analysis
allowed us to classify these neurons without a priori knowledge
of the number of groups. Our analysis yielded seven distinct
clusters into which PrS neurons could be classified, consistent
with our empirical observations. This technique further assists
in the uncovering of data structures and the formulation of
more objective schema for characterization and classification
of neuronal subtypes in brain areas of interest (Cauli et al.
2000; Garrido-Sanabria et al. 2007; Halabisky et al. 2006;
Nowak et al. 2003; Pennartz et al. 1998). To assess changes in
intrinsic properties of these neurons in TLE, we remeasured
these parameters in neurons from epileptic animals and repeated the cluster analysis with neurons from both groups. We
reasoned that if intrinsic properties of neurons in the epileptic
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logical classification of neocortical neurons (Abbasi and Kumar 2013;
Cauli et al. 2000; Halabisky et al. 2006; Pilli et al. 2012).
Parameters addressing the adaptive properties of cells were gathered from a 1-s long window of action potential trains evoked by a
100-pA depolarizing current pulse. The instantaneous firing frequencies between the first two spikes (finitial), 200 ms after the beginning
of the discharge (f200), and at the end of the window (ffinal) were
measured. Early and late spike frequency adaptation was calculated
using (finitial ⫺ f200)/finitial and (f200 ⫺ ffinal)/finitial, respectively.
Instantaneous and steady-state firing frequencies represented averages
of the instantaneous firing frequencies of 10 action potentials at the
beginning of a train and from a section where the discharge had
reached steady state. Sag ratio, a measure of sag conductance (Gs),
was determined as [(Vpeak ⫺ Vss)/Vpeak] ⫻ 100, where Vpeak and Vss
are measurements of voltage immediately after a negative 100-pA,
600-ms hyperpolarizing current pulse. Vpeak is voltage measured at the
negative-most point in the 600-ms window, and Vss is the measurement at the steady state after the initial voltage excursion. Accommodative hump was determined by the difference between the peak of the
smallest and the peak of the largest action potential following a
depolarizing current pulse and was used to quantify the tendency of
some cells to display a characteristic initial reduction followed by an
increase in action potential amplitude. Delay, a measure of the time it
takes for the cell to fire the first action potential from the onset of
current injection, was used to characterize a group of cells recorded
from in control animals that fired significantly later than other cells.
EXCITABILITY OF PRESUBICULAR NEURONS IN TLE
group were unaltered, they would aggregate/cluster with neurons with similar attributes from the control group. We found
that neurons from epileptic animals generally clustered with
only three of seven cell types encountered in the control group,
A
spontaneous
recurrent seizures
male SD rat
40 hr/wk video
monitoring
CONTROL
LATENT PERIOD
1 - 3 weeks
DOB
electrophysiology
EPILEPTIC
3 - 8 days
status
pilocarpine
treatment epilepticus
A
ME
LE
A
Par
AB
PrS
Su
CA1
1 mm
B
L1
V/VI
L2
III
L3
II
L4
control
250 um
2
1
C
epileptic
L5/6
8
6
5
3
D
4
10
9
7
2000
squared eucliden distance
1750
RS Cells in Epileptic Animals Fire Multiple Action
Potentials
2000
1500
1000
500
0
1
750
5
10
RS
IB
Hyperexcitability of PrS neurons was assessed based on
their propensity for firing multiple synaptically evoked action
CONTROL
IR
LS
Stu
FA
SS
TLE
500
IR
RS
FA
LS
250
FA
multi-spiking cells
SS
Stu
0
E
600
600
400
squared euclidean distance
200
0
CONTROL
1
5
TLE
10
IR
RS
IB
IR
RS
400
IR
200
0
namely the irregular-spiking (IR), fast-adapting (FA), and
regular-spiking (RS) cell types.
We carried out two rounds of cluster analysis. As with our
original characterization of PrS neurons in control animals, the
first round of clustering segregated all cells into five groups
(Fig. 1D), four of which [FA, late-spiking (LS), stuttering
(Stu), and single-spiking (SS)] had distinct firing profiles that
fit with our empirical classification, and one group, consisting
of multiple-spiking cells, with high intergroup variability that
deviated from our empirical classification. In the first round of
clustering, FA cells from the epileptic group clustered with
cells of the same type from the control group; IR and RS cells
from the epileptic group clustered with cells of the same type
from the control group in the multispiking cell category.
Introducing cells from the epileptic group did not alter clustering of LS, Stu, and SS cell types encountered only in the
control group. A second round of clustering (Fig. 1E), limited
to just the multispiking cells, resulted in further classification
of the remaining three cell types, RS, IR, and initially bursting
(IB) cell types, of which IB cells were encountered only in the
control group. IR cells from control and epileptic groups
clustered together but not with the RS or IB cell groups.
Likewise, with the exception of few cells, RS cells from
control and epileptic groups clustered into one group that was
well segregated from the IB or IR cell groups. These data
suggest that the cell-type-specific classification established in
control animals is generally applicable to cell types encountered in the epileptic animals. The following sections describe
the alterations in physiology observed in the three cell types
encountered in the epileptic animals.
RS
IB
Fig. 1. The presubicular slice preparation used in our study. A: schematic
outlining the various steps involved in bringing up the pilocarpine model of
temporal lobe epilepsy (TLE). Low-powered images of Nissl-stained sections
from control (left) and epileptic (right) animals, identifying the major anatomical landmarks: presubiculum (PrS), parasubiculum (Par), subiculum (Sub),
medial entorhinal area (MEA), dentate gyrus (DG), hippocampal CA1, and
lateral entorhinal area (LEA). Red arrows indicate the mediolateral extent of
LII in PrS (region demarcated by red dashed lines). B: high-powered image of
PrS (left) highlighting LII (white arrows) and various lamina (indicated by
Roman numerals) and MEA (right) from control (Œ) and epileptic () animals
with demarcated regions of interest from A. C: electrophysiological parameters
used for cell classification: 1) early spike frequency adaption (%), 2) late spike
frequency adaptation (%), 3) instantaneous firing frequency (Hz), 4) steadystate firing frequency (Hz), 5) sag ratio (%), 6) measure of input resistance
(M⍀), 7) delay to first action potential (ms), 8) action potential half-width
(ms), 9) spike-firing threshold (mV), and 10) afterhyperpolarization (mV).
D–E: classification of all neurons (D) and multispiking cells (E) in LII and III
of PrS using unsupervised hierarchical cluster analysis based on the above
parameters (Table 1 and Fig. 1C). Intersection of dendrogram branches with
the x-axis represents individual cells, and the y-axis represents the squared
Euclidean distances between group centroids at each branch point (longer
vertical lines indicate greater dissimilarity). Dashed lines indicate number of
groups as determined by the Thorndike method (see MATERIALS AND METHODS).
Inset: Thorndike method suggests 5 groups in a scree plot; x-axis: clustering
stages, y-axis: squared Euclidean distance between group centroids. Individual
cells from control and epileptic animals are color coded according to group
membership (control only cell types are not colored). Cell types: irregularspiking (IR), regular-spiking (RS), initially bursting (IB), late-spiking (LS),
stuttering (Stu), fast-adapting (FA), and single-spiking (SS).
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b
DG
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EXCITABILITY OF PRESUBICULAR NEURONS IN TLE
Table 1. Action potential waveform properties of PrS neurons
Electrophysiological Parameter
IR-Con
(n ⫽ 15)
IR-Epi
(n ⫽ 5)
⫺69 ⫾ 2
⫺41 ⫾ 1
60 ⫾ 7
Late spike frequency adaptation, %
25 ⫾ 5
18 ⫾ 5
Delay, ms
Accommodative hump, mV
Half-width, ms
35 ⫾ 3
1.4 ⫾ 0.4
2.8 ⫾ 0.2
32 ⫾ 8
1.0 ⫾ 0.2
2.2 ⫾ 0.1
Input resistance, M⍀
264 ⫾ 22
196 ⫾ 16
Sag ratio, %
1.8 ⫾ 0.2
3.5 ⫾ 1.8
Maximum frequency, Hz
13 ⫾ 1.8
22 ⫾ 4.1
Instantaneous firing frequency, Hz
Steady-state firing frequency, Hz
SD of steady-state firing frequency, Hz
9 ⫾ 1.0
4 ⫾ 0.5
1.7 ⫾ 0.2
13 ⫾ 1.5
6 ⫾ 0.7
2.3 ⫾ 0.4
FA-Epi
(n ⫽ 4)
⫺62 ⫾ 1
⫺63 ⫾ 3
⫺40 ⫾ 1
⫺42 ⫾ 2
57 ⫾ 4
61 ⫾ 3
RS-Epi ⬎⬎ RS-Con
n/d
n/d
RS-Con ⬎ RS-Epi
13 ⫾ 2
16 ⫾ 7
3.3 ⫾ 0.9
0.8 ⫾ 0.1
2.5 ⫾ 0.3
2.1 ⫾ 0.3
RS-Con ⬎⬎ RS-Epi
341 ⫾ 35
166 ⫾ 17
FA-Con ⬎ FA-Epi
1.9 ⫾ 0.6
0.7 ⫾ 0.4
RS-Epi ⬎⬎ RS-Con
39 ⫾ 4.5
24 ⫾ 4.4
RS-Epi ⬎ RS-Con
24 ⫾ 3.2
15 ⫾ 2.7
n/d
n/d
n/d
n/d
RS-Con
(n ⫽ 116)
RS-Epi
(n ⫽ 43)
⫺66 ⫾ 1
⫺40 ⫾ 1
40 ⫾ 2
⫺66 ⫾ 1
⫺39 ⫾ 1
51 ⫾ 2
8⫾1
6⫾1
25 ⫾ 1
2.2 ⫾ 0.2
2.2 ⫾ 0.1
28 ⫾ 7
2.3 ⫾ 0.3
1.8⫾ 0.1
330 ⫾ 8
306 ⫾ 15
2.2 ⫾ 0.2
4.7 ⫾ 0.8
35 ⫾ 1.7
43 ⫾ 3.1
24 ⫾ 0.9
16 ⫾ 0.7
1.1 ⫾ 0.1
26 ⫾ 1.4
17 ⫾ 0.9
1.1 ⫾ 0.1
Values represent means ⫾ SE; n, number of cells. PrS, presubiculum; Con, control group; Epi, epileptic group; RS, regular-spiking cell; IR, irregular-spiking
cell; FA, fast-adapting cell. Electrophysiological parameters were measured as described in MATERIALS AND METHODS. Significant differences between Epi and
Con groups are italicized. ⬎, significantly greater with P ⬍ 0.05, t-test; ⬎⬎, significantly greater, with P ⬍ 0.01, t-test; n/d, not determined because number
of action potentials was insufficient for reliable analysis.
potentials in response to increasing stimulus intensity, expressed as multiples of T, the threshold for evoking a single
action potential (Fig. 2). IR (n ⫽ 3 control and n ⫽ 3 epileptic),
FA (n ⫽ 5 control and n ⫽ 4 epileptic), and RS (n ⫽ 38 control
and n ⫽ 15 epileptic) cells from control and epileptic groups
were compared following whole cell current-clamp recording
of neurons from LII-III of PrS (Fig. 2A). We found that IR and
FA cells from epileptic animals were indistinguishable from
corresponding cell types in the control group based either on
the mean number of action potential evoked and/or the area
under the composite excitatory postsynaptic potential (EPSP;
P ⬎ 0.2 for both; Fig. 2B). RS cells from epileptic animals, on
the other hand, were found to be hyperexcitable, firing multiple
action potentials in response to even modest current increments. Both the number of action potentials evoked in response
to increasing stimulus intensity and area under the composite
EPSP were significantly increased in epileptic animals compared with controls (P ⬍ 0.001 at the indicated ⫻T intensities;
Fig. 2B) despite comparable T (control: 50 ⫾ 11, epileptic:
42 ⫾ 5 ␮A; P ⫽ 0.6) values. These data suggest that the
propensity of RS cells, but not IR or FA cells, to fire multiple
action potentials is significantly enhanced under epileptic conditions. We next measured changes in the excitatory and inhibitory
synaptic drive in these cell types under epileptic conditions.
IR Cells are Unaffected in Epileptic Animals
IR (Fig. 3, A1 and A2) cells display an action potential
discharge profile characterized by the firing of irregularly
spaced action potentials in response to current injections or
depolarization (Fig. 3B). In control animals, IR cells (n ⫽ 15)
were found exclusively in LII of PrS and comprised 8% of the
total sampled population. In epileptic animals, IR cells (n ⫽ 5)
were again found to be restricted to LII (Fig. 3A) and accounted for 10% of the total sampled population. Currentclamp recordings revealed no significant changes in electrophysiological parameters associated with action potential
waveform between the control and epileptic populations (Table
1). IR cells from both groups had characteristically larger
standard deviation of the steady-state firing frequency, higher
late spike frequency adaptation, and lower instantaneous and
steady-state firing frequencies relative to other cell types.
Consistent with these observations, IR cell excitability, as
assessed by the number of action potentials fired as a function
of increasing intensity of current injection, was comparable
between neurons from the control and epileptic groups (P ⬎
0.1). Voltage-clamp data revealed no significant alterations in
frequency and amplitude of either spontaneous or miniature
EPSCs and IPSCs (Fig. 3, C–F, and Table 2). Together, these
data indicate that IR cells are less affected by TLE relative to
other cell types in our sampling of PrS neurons from control
and epileptic animals.
Excitatory Synaptic Drive in FA Cells Is Enhanced During
TLE
FA (Fig. 4, A1 and A2) cells respond to current injection
or depolarization by firing action potentials at regularly
spaced intervals for a brief period of time before becoming
quiescent for the remainder of cell depolarization (Fig. 4B).
The discharge profile for FA cells was generally unaffected
by increased current injection. In both control and epileptic
animals, FA cells were restricted to LII of PrS, accounting
for 6% (n ⫽ 12) and 8% (n ⫽ 4) of total sampled cell types
in PrS, respectively. Current-clamp data revealed that with
the exception of a significant reduction in input resistance in
epileptic animals (341 ⫾ 35 vs. 166 ⫾ 17 M⍀; P ⬍ 0.05),
intrinsic firing properties remain unchanged between the
control and epileptic groups (Table 1). FA cell excitability,
as assessed by the number of action potentials fired as a
function of increasing intensity of current injection, was
qualitatively different from IR cells, but comparable between neurons from the control and epileptic groups (P ⬎
0.3). Voltage-clamp data revealed that the average fre-
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⫺69 ⫾ 1
⫺41 ⫾ 1
47 ⫾ 6
Resting membrane potential, mV
Spike firing threshold, mV
Early spike frequency adaptation, %
FA-Con
(n ⫽ 12)
EXCITABILITY OF PRESUBICULAR NEURONS IN TLE
RS Cells Are Less Inhibited Under Epileptic Conditions
control - RS
A
T
2T
4T
R
.
S
-65mV
epileptic - RS
epileptic - IR
20mV
200ms
mean evoked
action potentials
3
RS Control
RS Epileptic
IR Epileptic
3.5
***
2
area under EPSP
(mV * ms, x1000)
-69mV
2.5
***
***
***
***
1.5
1
0.5
T
2T
4T
stimulus intensity
T
2T
4T
stimulus intensity
Fig. 2. Hyperexcitability of RS cells in TLE. A: representative examples of
action potentials evoked in RS and IR cells under the indicated conditions in
response to stimulation at threshold (T) and increasing multiples (⫻2 and ⫻4)
of T. Resting membrane potential for cells is indicated along with placement
of stimulating (S) and recording (R) electrode in brain slices (inset). B: scatter
plots for mean number of action potentials evoked and area under the
composite excitatory postsynaptic potential (EPSP) in response to increasing
stimulus intensity for the indicated cell types. ***P ⬍ 0.001, t-test.
quency of sEPSCs in FA cells from epileptic rats was
⬃215% of control rats (1.3 ⫾ 0.3 vs. 2.8 ⫾ 0.6 Hz; P ⬍
0.05; Fig. 4C and Table 2). The increase in sEPSCs frequency may have resulted from increased activity of presynaptic excitatory circuits as no significant changes in
frequency or amplitude of mEPSCs (P ⫽ 0.1 and 0.8,
respectively) were observed. The frequency and amplitude
of sIPSCs and mIPSCs were unaffected (frequency: P ⫽ 0.2
and 0.1; amplitude: P ⫽ 0.4 and 0.4; Fig. 4D and Table 2)
in FA cells suggesting that synaptic inhibitory drive to these
neurons is not compromised under epileptic conditions.
Overall, these data suggest that FA cells are not hyperexcitable in the epileptic animals, but an enhanced excitatory
synaptic drive is seen under these conditions.
RS (Fig. 5, A1 and A2) cells are characterized by sustained
firing of regularly spaced action potentials in response to
current injections or depolarization (Fig. 5B). RS cells were the
predominant cell type in the PrS found in both LII and LIII of
control and epileptic animals, comprising 62% (n ⫽ 116) of the
sampled population in control animals and 82% (n ⫽ 43) in
epileptic animals. Current-clamp data from RS cells in control
and epileptic animals revealed similar resting membrane potentials and input resistances (Table 1). RS cells were the only
group of PrS neurons in epileptic animals that showed significant changes in action potential discharge profiles resulting
from current injection or cell depolarization; half-widths of
individual action potentials were reduced (control: 2.2 ⫾ 0.1;
epileptic: 1.8 ⫾ 0.1 ms; P ⬍ 0.05), maximum firing frequency
of action potentials was increased (control: 35 ⫾ 1.7; epileptic:
43 ⫾ 3.1 Hz; P ⬍ 0.05), early spike-frequency adaptation was
increased (control: 40 ⫾ 2; epileptic: 51 ⫾ 2%; P ⬍ 0.001),
while late adaptation was decreased (control: 8 ⫾ 1; epileptic:
6 ⫾ 1%; P ⬍ 0.05). These data suggest that RS cells in
epileptic animals have an increased initial firing frequency in
response to depolarization and a faster firing frequency adaption, which presumably enables them to reach steady-state
firing frequency sooner compared with RS cells in control
animals. Consistent with these observations, RS cell excitability, as assessed by the number of action potentials fired as a
function of increasing intensity of current injection, was enhanced in neurons from the epileptic groups compared with
control, although these differences did not reach the level of
statistical significance (P ⬎ 0.1, t-test). Additionally, RS cells
in epileptic animals had an increased sag ratio (control: 2.2 ⫾
0.2, epileptic: 4.7 ⫾ 0.8%; P ⬍ 0.001; Fig. 5B), a measure of
the hyperpolarization-activated Ih current. We noted that RS
cells in control animals showed little to no sag in response to
hyperpolarizing current injection compared with RS cells in
epileptic animals that displayed a more noticeable sag conductance (Fig. 5B). Although the significance of somatic assessments of sag in relation to hyperexcitability of neurons in TLE
is unclear (Notomi and Shigemoto 2004; Shah et al. 2004) and
possibly requires a direct assessment of Ih, these differences
were clearly manifest in RS cells from the control and epileptic
groups. It should be noted that the role of Ih (epileptogenic vs.
compensatory) and the underlying hyperpolarization-activated
cation nonselective 1 (HCN1) channels in mediating seizure
threshold is still under debate (Chen et al. 2001; Huang et al.
2009) given that TLE differentially affects their expression at
the soma and dendrite (Shah et al. 2004; Shin et al. 2008) and
Ih currents can vary depending on the time of measurement
following status (Jung et al. 2007).
RS cells in epileptic animals had significantly reduced sEPSCs frequency (control: 2.3 ⫾ 0.2; epileptic: 1.2 ⫾ 0.1 Hz;
P ⬍ 0.001) and amplitude (control: 19 ⫾ 0.7; epileptic: 15 ⫾
1.3 pA; P ⬍ 0.005; Fig. 5, C–F and Table 2). Decreases in
sEPSC frequency could potentially arise from loss of excitatory synapses, reductions in probability of neurotransmitter
release, and/or reduced activity of presynaptic neurons. However, the frequency and amplitude of mEPSCs in RS cells in
epileptic animals were unaltered compared with controls (P ⬎
0.1 for both; Fig. 6A and Table 2), ruling out the first two
alternatives in favor of a decreased excitatory drive to these
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-68mV
B
2893
2894
EXCITABILITY OF PRESUBICULAR NEURONS IN TLE
A1
LIII
A2
LIII
LII
LII
LI
pia
LI
Irregular Spiking (IR)
100 pA
100 pA
-100 pA
B
20 mV
20 mV
200 ms
375 ms
100 ms
control
epileptic
20
# APs
15
10
5
-66 mV
-70 mV
0
0
50
100
150
200
Current (pA)
C
control
D
sEPSCs (aCSF)
sIPSCs
20pA
10s
170ms
100pA
E
mEPSCs (+TTX)
F
mIPSCs
neurons under epileptic conditions. Interestingly, inhibitory
drive to RS cells was also reduced in the epileptic animals
(sIPSC frequency: 0.7 ⫾ 0.1 vs. 0.4 ⫾ 0.1 Hz; P ⬍ 0.05; sIPSC
amplitude: 45 ⫾ 1.6 to 27 ⫾ 1 pA; P ⬍ 0.001; Figs. 5, D–F,
and 6A and Table 2) and mIPSCs frequency and amplitude in
RS neurons from epileptic animals were 174% (0.23 ⫾ 0.04 vs.
0.4 ⫾ 0.04; P ⬍ 0.05; Figs. 5F and 6A) and 80% (30 ⫾ 2 vs.
24 ⫾ 1 pA; P ⬍ 0.005) of their values in control, respectively.
An increase in mIPSC frequency suggests that the probability
of neurotransmitter release from GABAergic inhibitory neurons is increased under epileptic conditions, and reductions in
mIPSC amplitude indicate a concomitant decrease in number
of GABAergic receptors expressed by these neurons and/or
alterations in receptor function.
Inclusion of TTX (1 ␮M) in the perfusate enables assessment of the proportion of synaptic events that are action
potential dependent. We found that TTX significantly reduced
both the frequency and amplitude of EPSCs and IPSCs in RS
cells from control animals but only affected the amplitude (not
frequency, P ⬎ 0.6 paired t-test; Fig. 6B, Table 2) of the PSCs
in epileptic animals. Thus, in epileptic animals, synaptic events
were predominantly action potential independent. This suggests that the RS cells are driven less by presynaptic neurons in
epileptic
10s
170ms
epileptic animals than in the controls, presumable because
presynaptic neurons are less active under these conditions. The
overall shift in synaptic transmission from action potential
dependent to action potential independent in RS cells under
epileptic conditions is reflected in the observed reductions in
mean amplitude of spontaneous events as action potentialdependent events are typically larger in amplitude than action
potential-independent events.
DISCUSSION
The present study characterizes the alterations in physiological properties of neurons in superficial LII-III of PrS in a rat
model of TLE. Our principal findings are the following: 1) only
a subset of PrS neurons (1 of 3 major cell types) undergoes
alterations in action potential discharge profiles and excitatory
and inhibitory synaptic drive, making TLE-related changes in
this structure cell type specific; and 2) RS cells are hyperexcitable in TLE. This is the predominant cell type in PrS found
in both control and epileptic animals and identified previously
as a class of projection neurons with the potential to entrain
downstream structures, particularly the MEA, in TLE (Abbasi
and Kumar 2013).
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Fig. 3. IR cells. A1: representative photomicrograph
captured on a confocal microscope of a biocytinlabeled IR neuron in LII of PrS from an epileptic rat.
Roman numerals (I-III) indicate lamina and scale
bar ⫽ 100 ␮m. A2: neurolucida reconstruction of the
IR cell in A1 showing laminar location of somata and
dendritic morphology. B: representative examples of
action potential discharge in IR cells from control and
epileptic animals. Action potential (AP) waveforms in
response to sustained current injections of 100 pA (1to 10-s duration; left) and hyper- and depolarizing
current pulses (⫾100 pA, 600 ms duration; middle)
from resting membrane potential (Vm). C–F: excitatory and inhibitory synaptic drive to IR cells from
control and epileptic animals, measured using voltage
clamp. Graph of the mean number of action potentials
as a function of depolarizing current injection from
control and epileptic neurons (0 –200 pA in 50-pA
increments, 600-ms duration; right). C: trace (1 min
long) of spontaneous excitatory postsynaptic currents
[sEPSCs, inward events recorded at ⫺70-mV holding
potential in artificial cerebrospinal fluid (aCSF)] recorded from IR cells in control (left) and epileptic
animals (right). In this and all subsequent figures,
embedded insets offer an expanded view of the indicated portions of traces (dotted lines). D: spontaneous
inhibitory postsynaptic currents (sIPSCs, outward
events recorded at 0 mV in aCSF). E: miniature
excitatory postsynaptic currents [mEPSCs, inward
events recorded at ⫺70 mV in the presence of 1 ␮M
tetrodotoxin (TTX)]. F: miniature inhibitory postsynaptic currents (mIPSCs, outward events recorded at 0
mV in TTX).
EXCITABILITY OF PRESUBICULAR NEURONS IN TLE
A1
A2
LIII
LIII
LII
LII
LI
pia
LI
100 μm
Fast Adapting (FA)
100 pA
100 pA
-100 pA
B
20 mV
20 mV
200 ms
280 ms
100 ms
control
epileptic
20
# APs
10
5
-66 mV
-63 mV
0
0
50
100
150
Current (pA)
D
control
20pA
10s
170ms
sEPSCs (aCSF)
epileptic
sIPSCs
200
Fig. 4. FA cells. A1: representative photomicrograph
captured on a confocal microscope of a biocytinlabeled FA neuron in LII of PrS from an epileptic rat.
Roman numerals (I-III) indicate lamina and scale
bar ⫽ 100 ␮m. A2: neurolucida reconstruction of the
FA cell in A1 showing laminar location of somata and
dendritic morphology. B: representative examples of
action potential discharge in FA cells from control and
epileptic animals. Action potential waveforms in response to sustained current injections of 100 pA (1- to
10-s duration; left) and hyper- and depolarizing current
pulses (⫾100 pA, 600-ms duration; middle) from resting membrane potential (Vm). C–F: excitatory and
inhibitory synaptic drive to FA cells from control and
epileptic animals, measured using voltage clamp. Graph
of the mean number of action potentials as a function of
depolarizing current injection from control and epileptic neurons (0 –200 pA in 50-pA increments, 600-ms
duration; right). C: trace (1-min long) of sEPSCs
(inward events recorded at ⫺70-mV holding potential
in aCSF) recorded from FA cells in control (left) and
epileptic animals (right). Embedded insets offer an
expanded view of the indicated portions of traces
(dotted lines). D: sIPSCs (outward events recorded at 0
mV in aCSF) E: mEPSCs (inward events recorded at
⫺70 mV in the presence of 1 ␮M TTX). F: mIPSCs
(outward events recorded at 0 mV in TTX).
mEPSCs (+TTX)
E
100pA
F
10s
170ms
mIPSCs
Compared with RS cells, IR and FA cells in the PrS
appeared to be relatively unaffected in epileptic animals based
on intrinsic firing properties and synaptic drive with the exceptions of an increase in frequency of sEPSCs noted in FA
cells along with a decrease in input resistance, similar to that
reported for subicular pyramidal neurons in pilocarpine-treated
rats (Knopp et al. 2005; Wellmer et al. 2002). Note that these
observations for IR and FA cells are from a small number of
neurons (n ⬍ 10), owing to the relatively small proportion of
these neuronal types in our sampled population of neurons
from epileptic animals. RS cells in epileptic animals, on the
other hand, displayed changes in action potential waveform
properties supporting higher rates of action potential discharge
and enhanced early spike frequency adaptation. Although we
did not directly assess underlying mechanisms, previous studies have attributed similar changes in TLE to altered gating,
increased levels of Na⫹ channel expression (Hargus et al.
2011), and modifications to Ca2⫹-activated BK class of K⫹
channels (Brenner et al. 2005).
RS cells from epileptic animals had significantly reduced
excitatory and inhibitory synaptic drive, and data from voltageclamp experiments suggest reduced activity in both excitatory
and inhibitory circuits presynaptic to the recorded RS cells. An
overall reduction in synaptic drive to RS cells conforms to a
model in which reductions in excitatory drive to these neurons
may also manifest in presynaptic GABAergic interneurons that
are capable of providing feed-forward inhibition keeping their
excitability in check (Fig. 7). Future work includes identification of GABAergic neurons that target RS cells and the direct
assessment of synaptic drive to these neurons under control and
epileptic conditions. Alternatively, GABAergic interneurons
providing feedback inhibition might also be affected, although
this appears unlikely because RS cells are hyperexcitable in
epileptic animals. Although the precise sources of excitation to
RS cells remain unknown, previous studies examining TLErelated alterations in neuronal populations suggest neuron loss
in a number of structures that project to the PrS, including
retrosplenial granular cortex, thalamus, and subiculum. It is
likely that alterations in the synaptic drive to RS cells that were
observed in the epileptic animals arise as a consequence of
neuron loss in these structures (Cardoso et al. 2008; Chen and
Buckmaster 2005; Covolan and Mello 2000; Dube et al. 2001;
Scholl et al. 2013).
The RS cells of PrS are not the only neurons found to be
hyperexcitable in epileptic animals; previous studies of TLE
using animal models have also reported hyperexcitability of
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15
C
2895
2896
EXCITABILITY OF PRESUBICULAR NEURONS IN TLE
A1
A2
Regular Spiking (RS)
LII
LII
LII
LI
pia
LI
pia
LI
100 μm
100 pA
100 pA
-100 pA
B
20 mV
20 mV
200 ms
270 ms
100 ms
control
epileptic
20
# APs
15
10
5
-67 mV
-63 mV
0
0
50
100
150
200
Current (pA)
C
D
E
20pA
control
sEPSCs (aCSF)
epileptic
10s
170ms
sIPSCs
mEPSCs (+TTX)
100pA
F
MEA neurons (Bear et al. 1996; Scharfman et al. 1998; Tolner
et al. 2007) mediated by modifications to both synaptic and
intrinsic properties (Hargus et al. 2011; Kumar et al. 2007;
Shah et al. 2004). Alterations in synaptic inhibition, in particular, have been identified as the major underlying cause for
pathophysiological alterations associated with TLE (El-Hassar
et al. 2007; Kobayashi et al. 2003; Kumar and Buckmaster
2006; Obenaus et al. 1993), and suppression of network inhibition through pharmacological blockade of GABAA synaptic
transmission in either the MEA or PrS was found sufficient to
promote hyperexcitability (Funahashi et al. 1999; Menendez de
la Prida and Pozo 2002), suggesting similar pathophysiological
processes underlying hyperexcitability in the two regions.
Cell Loss and Classification of Neurons in the Epileptic PrS
We previously identified seven electrophysiologically distinct cell types in LII-III of PrS in normal animals (Abbasi and
Kumar 2013). However, our assessment of the PrS under
mIPSCs
10s
170ms
epileptic conditions yielded only three of these cell types (FA,
IR, and RS). PrS cell types that were not encountered in
epileptic animals included IB and Stu cells, a class of putative
inhibitory interneurons (Abbasi and Kumar 2013). One explanation for this discrepancy is the greater vulnerability of certain
neuronal populations to perish during TLE. Indeed, previous
studies have identified differences in the susceptibility of PrS
neurons including reductions in the number of parvalbumin
and calretinin-positive GABAergic interneurons, reductions in
total cell counts, and increased labeling of markers of neuronal
degeneration (Cardoso et al. 2011; Drexel et al. 2011; Scholl et
al. 2013; van Vliet et al. 2004). The absence of IB neurons
from our sample of the epileptic PrS is reminiscent of a study
in the subiculum of pilocarpine-treated, chronically epileptic
rats that reported drastic reductions in the population of bursting neurons compared with controls (Knopp et al. 2005),
although other studies have reported an upregulation of bursting neurons in the subiculum following pilocarpine-induced
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Fig. 5. RS cells. A1: representative photomicrograph captured on a confocal microscope of biocytin-labeled RS neurons in LII and LIII of PrS from
an epileptic rat. Roman numerals (I-III) indicate
lamina and scale bar ⫽ 100 ␮m. A2: beurolucida
reconstruction of RS cells in A1 showing laminar
location of somata and dendritic morphology. B:
representative examples of action potential discharge in RS cells from control and epileptic animals. Action potential waveforms in response to
sustained current injections of 100 pA (1- to 10-s
duration; left) and hyper- and depolarizing current
pulses (⫾100 pA, 600-ms duration; middle) from
resting membrane potential (Vm). C–F: excitatory
and inhibitory synaptic drive to RS cells from control and epileptic animals, measured using voltage
clamp. Graph of the mean number of action potentials as a function of depolarizing current injection
from control and epileptic neurons (0 –200 pA in
50-pA increments, 600-ms duration; right). C: trace
(1-min long) of sEPSCs (inward events recorded at
⫺70-mV holding potential in aCSF) recorded from
RS cells in control (left) and epileptic animals
(right). Embedded insets offer an expanded view of
the indicated portions of traces (dotted lines). D:
sIPSCs (outward events recorded at 0 mV in aCSF)
E: mEPSCs (inward events recorded at ⫺70 mV in
the presence of 1 ␮M TTX). F: mIPSCs (outward
events recorded at 0 mV in TTX).
LIII
LIII
LIII
EXCITABILITY OF PRESUBICULAR NEURONS IN TLE
mIPSC
RS
sEPSC
mEPSC
sIPSC
mIPSC
spontaneous (aCSF)
miniature (aCSF + TTX)
3
3
2
2
***
ns
1
1
*
*
0
0
Control
Control
Epileptic
ns
Epileptic
30
Values represent means ⫾ SE. The total number of cells tested (n) is
indicated for each group, reported for spontaneous and miniature experiments,
respectively. Frequency is reported in hertz (Hz), and amplitude is reported in
picoamp (pA). Values are for spontaneous (s) and miniature (m) excitatory
postsynaptic current (EPSC) and inhibitory postsynaptic current (IPSC). *P ⬍
0.05, †P ⬍ 0.01, ‡P ⬍ 0.001, t-test.
50
25
40
20
15
**
***
30
**
20
10
Control
Control
Epileptic
Epileptic
Frequency
mEPSCs
mIPSCs
B
**
120
80
80
60
60
40
40
20
20
% in TTX
(aCSF = 100%)
100
0
*
120
100
control epileptic
0
control epileptic
Amplitude
120
120
ns
100
% in TTX
(aCSF = 100%)
status (Wellmer et al. 2002). It is plausible to attribute these
differences to the short- and long-term consequences of an
epileptogenic treatment (Kumar and Buckmaster 2006). Additionally, there might be differences in the effects of pilocarpine
treatment and TLE along the proximal-distal axis of the subiculum, with Knopp et al. (2005) recording preferentially in the
middle portion of the subiculum that projects to the PrS, in
contrast with Wellmer et al. (2002) whose recordings are in
proximal subiculum that does not to project to the PrS
(O’Reilly et al. 2013). It has been suggested that an increased
Ca2⫹ conductance, deemed important for bursting activity
(Jung et al. 2001), contributes to the vulnerability of these
neurons to excitotoxic cell death; a hypothesis also proposed
for explaining vulnerability of neuronal populations in the
MEA (Gloveli et al. 1999). Alternatively, differences in the
proportional distribution of cell types encountered in PrS
between control and epileptic groups might be related to
alterations in intrinsic properties of neurons that modify their
action potential discharge properties resulting in their reclassification. However, we consider this a remote possibility
because cells in PrS appear not be interconvertible between cell
types (Abbasi and Kumar 2013). The fact that two of the three
cell types encountered in epileptic animals (IR and FA cells)
were unperturbed from control conditions and that all cell types
from both groups clustered according to the classification
schema for control animal supports this assertion. Finally, we
acknowledge the possibility of not recording from certain cell
IPSCs
4
100
80
80
60
60
40
40
20
20
0
control epileptic
0
ns
control epileptic
Fig. 6. Measurements of synaptic activity in RS cells from control and epileptic
animals. A: changes to frequency and amplitude of excitatory and inhibitory
postsynaptic currents under the indicated conditions. Statistical significance
(voltage-clamp) between control and epileptic groups: *P ⬍ 0.05; **P ⬍ 0.01;
***P ⬍ 0.001, t-test; before and after of TTX. ⫹P ⬍ 0.05, ⫹⫹P ⬍ 0.01,
⫹⫹⫹P ⬍ 0.001, paired t-test. B: bar plots comparing s- and mEPSC frequency and amplitude under control and epileptic conditions (for each plot
100% ⫽ magnitude of the response in aCSF). *P ⬍ 0.05; **P ⬍ 0.01, t-test.
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sIPSC
4
++
mEPSC
EPSCs
A
++
FA
sEPSC
+++
mIPSC
+
sIPSC
n ⫽ 5; 5
1.8 ⫾ 0.5 Hz
13 ⫾ 1.1 pA
1.2 ⫾ 0.3 Hz
12 ⫾ 1.2 pA
0.3 ⫾ 0.1 Hz
31 ⫾ 3.2 pA
0.3 ⫾ 0.1 Hz
26 ⫾ 1.9 pA
n ⫽ 4; 4
2.8 ⫾ 0.6 Hz*
14 ⫾ 2.0 pA
1.2 ⫾ 0.3 Hz
13 ⫾ 2.4 pA
0.2 ⫾ 0.1 Hz
29 ⫾ 3.1 pA
0.2 ⫾ 0.1 Hz
26 ⫾ 2.0 pA
n ⫽ 39; 29
1.2 ⫾ 0.1 Hz‡
15 ⫾ 1.3 pA†
0.9 ⫾ 0.2 Hz
12 ⫾ 0.9 pA
0.4 ⫾ 0.1 Hz*
27 ⫾ 1.0 pA‡
0.4 ⫾ 0.1 Hz†
24 ⫾ 1.2 pA†
+++
mEPSC
n ⫽ 15; 5
2.0 ⫾ 0.3 Hz
17 ⫾ 1.3 pA
1.1 ⫾ 0.2 Hz
11 ⫾ 0.6 pA
0.4 ⫾ 0.2 Hz
34 ⫾ 2.0 pA
0.2 ⫾ 0.1 Hz
23 ⫾ 2.6 pA
n ⫽ 12; 5
1.3 ⫾ 0.3 Hz
19 ⫾ 1.9 pA
0.7 ⫾ 0.1 Hz
14 ⫾ 2.4 pA
0.4 ⫾ 0.1 Hz
37 ⫾ 4.8 pA
0.4 ⫾ 0.1 Hz
25 ⫾ 1.4 pA
n ⫽ 113; 37
2.3 ⫾ 0.2 Hz
19 ⫾ 0.7 pA
1.1 ⫾ 0.1 Hz
14 ⫾ 0.8 pA
0.7 ⫾ 0.1 Hz
45 ⫾ 1.6 pA
0.2 ⫾ 0.1 Hz
31 ⫾ 1.7 pA
+++
sEPSC
Epileptic
Frequency (Hz)
IR
Control
types on account of their limited representation in the control
population (SS, 5%; LS, 2%; Stu: 4%) leading to sampling
errors in the population of neurons sampled from epileptic
animals, although the absence of IB cells, is likely not attributable to errors in sampling because this cell type accounted for
as much as 13% of our control population.
Note on sufficiency of neurons sampled. The number of cells
sampled in our control and epileptic groups was adequate to
make inferences and conclusions about changes in their physiological properties. Thus getting more cells to increase ns in
any category would not only be cumbersome, but impractical,
Amplitude (pA)
Table 2. Summary of frequency and amplitude of PSCs in PrS
neurons from control and epileptic groups
2897
2898
EXCITABILITY OF PRESUBICULAR NEURONS IN TLE
epileptic
control
direct
excitation
?
?
feed-forward
inhibition
RS
normal
hyperexcitable
pyramidal neuron
excitatory synaptic input
inhibitory synaptic input
normal synaptic drive
decreased synaptic drive
Fig. 7. A summary of possible changes in excitatory and inhibitory synaptic
drive rendering RS cells in the PrS hyperexcitable under epileptic conditions.
The brake analogy refers to synaptic inhibition of RS cells. Open questions (?)
include the identity of GABAergic neurons that target RS cells and the direct
assessment of synaptic drive to these neurons under control and epileptic
conditions.
given the difficulty of “searching” for particular cell types in
brain tissue under DIC optics and that doing so might also
preclude the obtaining of an unbiased random sampling of
neurons from the two populations for comparative purposes.
Given that certain cell types account for only a small percentage of all sampled neurons in PrS, boosting ns might only
minimally affect statistical inferences and may not resolve the
issue of missing cell types in the epileptic sample. For instance,
IB cells accounted for 13% of total sampled neurons in control
animals, a significantly larger portion of the sample, compared
with either IR (8%) or FA (6%) cells. The fact that we were
able to record from both IR and FA cells in epileptic tissue
(that accounted for 10 and 8% of sampled neurons from
epileptic animals, respectively) suggests that recording from IB
cells should have been possible. Increasing our sample size to
“uncover” IB cells in epileptic tissue could lead to the following scenarios: if we are unsuccessful in recording from these
cells, we can more confidently assert the vulnerability of this
PrS cell type in TLE. Alternatively, if we are successful in
recording from IB cells in any additional recordings, they most
likely will end up accounting for a smaller proportion of the
sample from epileptic animals than they did for controls,
thereby validating the vulnerability of IB cells in TLE. Both
these conclusions are not very different from those reached
through our experiments. On the other hand, Stu cells accounted for a smaller portion (4%) of our sampled control
population, and so it is conceivable that these cells were not
recorded from in epileptic animals because of errors in sampling and/or TLE-related changes in their distribution.
To the best of our knowledge this is a first attempt at
direct assessment of the physiological state of PrS neurons
in confirmed epileptic animals, with the potential to shed
light on the hyperexcitability of neurons in the neighboring
MEA and hippocampus. The PrS itself is capable of triggering hyperexcitability and mediating pathophysiological
events in neighboring MEA (Tolner et al. 2005, 2007; Zahn
et al. 2008) although details of the underlying mechanisms
have remained unknown. Our results pinpoint RS cells as
being hyperexcitable under epileptic conditions, a state
brought about by and/or exacerbated by reductions in synaptic inhibition. PrS projections to MEA have been worked
out anatomically (Caballero-Bleda and Witter 1993; Honda
et al. 2011; Honda and Ishizuka 2004; van Haeften et al.
1997) and characterized physiologically (Bartesaghi et al.
2005), and, more recently, shown to form functional connections with cells in all layers of MEA (Canto et al. 2012).
Our recent study of the PrS (Abbasi and Kumar 2013)
provided detailed electrophysiological characterization for
PrS projection neurons, including those targeting MEA,
which include RS cells. We have shown that projection
neurons in the PrS have bifurcating axons that seem to
concomitantly innervate the MEA and more distant target
structures (subiculum, retrosplenial granular cortex) via the
angular bundle, enabling them to potentially drive multiple
structures (Abbasi and Kumar 2013; Honda et al. 2011).
Additionally, it has been reported that PrS projections targeting MEA are preserved in epilepsy despite significant
loss of neurons in LIII of MEA (Tolner et al. 2005) (see
also, Fig. 1B). Our findings therefore implicate the PrS in
the entrainment of downstream structures including the
MEA under epileptic conditions and identify this parahippocampal structure as a potential locus for implementation
of cell-type-specific interventional/therapeutic strategies in
combating TLE.
ACKNOWLEDGMENTS
We are grateful to Richard L. Hyson and Frank Johnson for critically
reading the manuscript and for suggestions. We thank Max Richardson,
Tiffany Jacobson, John Gray, Mark Basista, and Jyotsna Pilli for contributions
to various aspects of this research endeavor. We are grateful to J. Barber, F.
Fletcher, and T. Tang of the support groups in the College of Medicine and
Department of Psychology at Florida State University for technical assistance.
GRANTS
This work was supported in part by grants from the Center for Research and
Creativity and College of Medicine at Florida State University and the
Epilepsy Foundation.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: S.A. and S.S.K. conception and design of research;
S.A. and S.S.K. performed experiments; S.A. and S.S.K. analyzed data; S.A.
and S.S.K. interpreted results of experiments; S.A. and S.S.K. prepared figures;
S.A. and S.S.K. drafted manuscript; S.A. and S.S.K. edited and revised
manuscript; S.A. and S.S.K. approved final version of manuscript.
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Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 17, 2017
GABAergic interneuron
Implications for Entrainment of Downstream Structures
EXCITABILITY OF PRESUBICULAR NEURONS IN TLE
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