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Electrophysiological and Morphological Characteriza-tion of Cells in Superficial Layers of Rat Presubiculum
Saad Abbasi and Sanjay S. Kumar*
Department of Biomedical Sciences, College of Medicine and Program in Neuroscience, Florida State University, Tallahassee,
Florida 32306-4300
ABSTRACTThe presubiculum (PrS) plays critical roles in spatial
information processing and memory consolidation and
has also been implicated in temporal lobe epileptogene-
sis. Despite its involvement in these processes, a basic
structure–function analysis of PrS cells remains far
from complete. To this end, we performed whole-cell
recording and biocytin labeling of PrS neurons in layer
(L)II and LIII to examine their electrophysiological and
morphological properties. We characterized the cell
types based on electrophysiological criteria, correlated
their gross morphology, and classified them into distinct
categories using unsupervised hierarchical cluster anal-
ysis. We identified seven distinct cell types: regular-
spiking (RS), irregular-spiking (IR), initially bursting (IB),
stuttering (Stu), single-spiking (SS), fast-adapting (FA),
and late-spiking (LS) cells, of which RS and IB cells
were common to LII and LIII, LS cells were specific to
LIII, and the remaining types were identified exclusively
in LII. Recorded neurons were either pyramidal or non-
pyramidal and, except for Stu cells, displayed spine-rich
dendrites. The RS, IB, and IR cells appeared to be pro-
jection neurons based on extension of their axons into
LIII of the medial entorhinal area (MEA) and/or angular
bundle. We conclude that LII and LIII of PrS are distinct
in their neuronal populations and together constitute a
more diverse population of neurons than previously sug-
gested. PrS neurons serve as major drivers of circuits
in superficial (LII–III) entorhinal cortex (ERC) and couple
neighboring structures through robust afferentation,
thereby substantiating the PrS’s critical role in the para-
hippocampal region. J. Comp. Neurol. 521: 3116–3132,
2013.
VC 2013 Wiley Periodicals, Inc.
INDEXING TERMS: parahippocampal region; subiculum; cell classification; physiology; morphology; entorhinal cortex
The presubiculum (PrS), defined by Brodmann as
area 27, is located between the subiculum and the par-
asubiculum and is cytoarchitectonically distinguished by
a densely packed pyramidal cell layer (LII). Layer II
combines with LIII to form the superficial layers, fol-
lowed by a cell-sparse LIV, which is juxtaposed with
what some believe to be the deep layers (LV–VI) of PrS
(see Fig. 1A,B). The PrS plays a pivotal role in the proc-
essing of spatial information (Boccara et al., 2010). In
the temporal lobe memory circuit, PrS serves as a path-
way for information from the hippocampus to enter the
entorhinal cortex (ERC; Bartesaghi et al., 2005). PrS
input arises from the retrosplenial and cingulate corti-
ces (Jones and Witter, 2007; Kononenko and Witter,
2012; van Groen and Wyss, 1990a,b), laterodorsal
nucleus of the thalamus (van Groen and Wyss, 1990a),
and subiculum (Kohler, 1985). In turn, the PrS sends
afferents to the subiculum, retrosplenial cortex (Van
Groen and Wyss, 2003), and thalamus as well as a
prominent projection to the medial entorhinal area
(MEA; Honda and Ishizuka, 2004). The projection to
MEA arises from all layers of PrS, although the projec-
tion from LIII is significantly more robust compared with
the other layers (Honda et al., 2011). These projections,
which selectively target LIII of MEA, and to a lesser
degree LII, provide both glutamatergic and
g-aminobutyric acid (GABA)-ergic input to the MEA (van
Haeften et al., 1997) and are of particular interest
because they provide a route by which activity in the
PrS can indirectly influence the neocortex, hippocam-
pus, and other parahippocampal regions via the ERC’s
Grant sponsor: Council on Research and Creativity and College ofMedicine at Florida State University; Grant sponsor: EpilepsyFoundation.
*CORRESPONDENCE TO: Sanjay S. Kumar, PhD, Department of Biomed-ical Sciences, College of Medicine, Suite 3300-B, Florida StateUniversity, 1115 West Call Street, Tallahassee, FL 32306-4300.E-mail: sanjay.kumar@med.fsu.edu
Received December 7, 2012; Revised May 2, 2013;Accepted for publication May 3, 2013.DOI 10.1002/cne.23365Published online May 16, 2013 in Wiley Online Library(wileyonlinelibrary.com)VC 2013 Wiley Periodicals, Inc.
3116 The Journal of Comparative Neurology | Research in Systems Neuroscience 521:3116–3132 (2013)
RESEARCH ARTICLE
vast network of connections (Canto et al., 2012; van
Strien et al., 2009).
Functionally, the importance of the PrS has been
demonstrated through its involvement in spatial learning
and temporal lobe epileptogenesis (TLE). The role of the
PrS in enabling an animal to form a spatial representa-
tion of its environment is highlighted by the presence
of functionally specific cell types in the form of head-
direction and grid cells (Boccara et al., 2010; Taube
et al., 1996). These cells send projections to the ERC
and other structures essential for navigational process-
ing (Moser and Moser, 2008). The PrS is an area of
interest in the study of TLE because of the projections
it sends to seizure-sensitive cells in LIII of MEA (Eid
et al., 1996), which have been implicated in the genera-
tion of epileptiform discharges during TLE (Tolner et al.,
2005, 2007) and in the characteristic loss of a vulnera-
ble population of cells in LIII observed in patients and
animal models of TLE (Du et al., 1993, 1995; Eid et al.,
2001; Kumar and Buckmaster, 2006).
Despite anatomical characterization that highlights
functional uniqueness of this region, basic electrophys-
iological properties of cells in the PrS are lacking, rela-
tive to neighboring ERC and hippocampus, and have
been addressed in only a handful of studies (Fricker
et al., 2009; Funahashi and Stewart, 1997a; Menendez
de la Prida et al., 2003; Simonnet et al., 2013). To fur-
ther our understanding of this region, we performed
whole-cell patch-clamp recordings from neurons in LII
and LIII of the PrS, combining electrophysiology with
morphology via biocytin labeling to characterize the
various cell types in the PrS. Our techniques for cell
characterization are similar to those used by other
researchers in characterizing cells of the neocortex
and other brain regions (Cauli et al., 2000; Kawaguchi
and Kubota, 1997; McCormick et al., 1985), thus mak-
ing it possible to compare neurons of PrS with those
of other regions. We used electrophysiological parame-
ters and unsupervised hierarchical cluster analysis to
classify cell types in PrS, similar to our recent classifi-
cation of cells in deep layers of the MEA (Pilli et al.,
2012).
LII and LIII of PrS are typically described as a com-
bined structure in the literature (superficial layers of
PrS) and have been described as containing a function-
ally homogeneous cell population possessing afferent
projections that show great overlap (Funahashi and
Stewart, 1997a; Honda and Ishizuka, 2004; Menendez
de la Prida et al., 2003; Simonnet et al., 2013). Our
study shows that both LII and LIII contain a physiologi-
cally diverse population of cell types with distinct lami-
nar distributions, patterns of projection, and functional
attributes.
MATERIALS AND METHODS
AnimalsSprague-Dawley rats (male) from postnatal days
40–75 were used in our study. All experiments were
carried out in accordance with the National Institute of
Health Guide for the care and use of laboratory animals
and were approved by the Florida State University Insti-
tutional Animal Care and Use Committee.
Slice preparation and electrophysiologyRats were deeply anesthetized with urethane (1.5 g/
kg ip) before being decapitated. Horizontal slices (350
lm), at the level of the hippocampus between 26.6
and 25.3 mm from Bregma (Paxinos and Watson,
2007), were prepared using a microslicer (Leica
VT1000S) in a chilled (4�C) low-Ca21, low-Na1 “cutting
solution” containing (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 equili-
brate in oxygenated artificial cerebrospinal fluid (aCSF)
containing (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 hour and subse-
quently at room temperature before being transferred
to the recording chamber.
Recordings were obtained at 32�C 6 1�C from neu-
rons in the PrS under Nomarski optics (Zeiss) using a
visualized infrared setup (Hamamatsu). Cell morphology
and location within the various lamina could be identi-
fied. Patch electrodes were pulled from borosilicate
glass (1.5 mm outer diameter, 0.75 mm inner diameter,
3–6 MX) and contained (in mM) 105 potassium gluco-
nate, 30 KCl, 10 HEPES, 10 phosphocreatine, 4 MgATP,
0.3 GTP, and 20 biocytin. Internal solution was adjusted
to a pH of 7.3 with KOH and an osmolarity of 300
mOsm. Slices were maintained in oxygenated (95%
O2–5% CO2) aCSF.
Postsynaptic currents and potentials were recorded
using a MultiClamp 700B amplifier and pCLAMP soft-
ware (Molecular Devices, Union City, CA), filtered at
1–2 kHz (10 kHz for current clamp), digitized at 10–20
kHz, and stored digitally. Series resistance was moni-
tored continuously, and those cells in which this param-
eter exceeded 20 MX 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 cur-
rent (1–10 seconds in duration) and 2) hyper- and
depolarizing current steps, 500 msec in duration and
50 pA in amplitude. The minimum current (pA) required
for evoking an action potential, averaged across cells in
Physiology and morphology of cells in presubiculum
The Journal of Comparative Neurology | Research in Systems Neuroscience 3117
each group of cells, regular-spiking (RS; 37 6 2),
irregular-spiking (IR; 60 6 5), initially bursting (IB; 35 6
4), stuttering (Stu; 58 6 10), single-spiking (SS; 70 6
8), fast-adapting (FA; 51 6 7), and late-spiking (LS; 76
6 11), was below 100 pA. Spontaneous excitatory
postsynaptic currents (sEPSCs) were obtained by hold-
ing the cell at 270 mV, and inhibitory postsynaptic cur-
rents (sIPSCs) were recorded at a holding potential of 0
mV, the reversal potential for glutamate. The postsynap-
tic current data, obtained from 2-min-long continuous
recordings, were analyzed with Mini Analysis (Synapto-
soft, Decatur, GA). The threshold for event detection
was set at 3 3 root mean square (RMS) noise level
(see Table 2). Average RMS noise levels were similar in
all groups (P 5 0.4). Software-detected events were
visually verified, and their frequency and amplitude
were measured.
Cluster analysisTo classify neurons of the PrS, an unsupervised hier-
archical cluster analysis was performed based on elec-
trophysiological parameters gathered from 177 neurons
in PrS. Clustering was performed to determine whether
the population of PrS neurons was heterogeneous and
also to establish a schema for their classification. Clus-
tering was carried out independently 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 multidi-
mensional space based on similarities of the electro-
physiological 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 neu-
rons, until the final stage, when a single cluster con-
tained all of the neurons. Ward’s method uses an
ANOVA approach in determining between-cluster distan-
ces. All variables were standardized prior to 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 minimiz-
ing 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 serve to indicate
salient differences between groups and cluster
numbers.
We considered nine electrophysiological parameters
in our analysis, all of which were gathered during
current-clamp recordings from each cell considered in
the analysis. These included 1) early spike frequency
adaptation, 2) late spike frequency adaptation, 3) delay,
4) accommodative hump, 5) sag ratio, 6) maximum fir-
ing 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 neu-
rons in the PrS along with consideration of additional
variables deemed most valuable in predicting group
membership in alternate studies of the electrophysio-
logical classification of neocortical neurons (Cauli et al.,
2000; Halabisky et al., 2006; Pilli et al., 2012).
Parameters addressing the adaptive properties of
cells were gathered from a 1-second-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 msec after the
beginning of the discharge (f200), and at the end of
the window (ffinal) were measured. Early and late
spike frequency adaptation were calculated using
(finitial 2 f200)/finitial and (f200 2 ffinal)/finitial, respec-
tively. Instantaneous and steady-state firing frequencies
represented averages of the instantaneous firing fre-
quencies 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 2 Vss]/
Vpeak) 3 100, where Vpeak and Vss are measurements of
voltage immediately after a negative 100-pA, 500-msec
hyperpolarizing current pulse; Vpeak is voltage measured
at the negative-most point in the 500-msec 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 charac-
teristic 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 charac-
terize a group of cells that fired significantly later than
other cells.
NeuN-biocytin immunohistochemistryTo 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
hours. After fixation, slices were stored in 30% ethylene
glycol and 25% glycerol in 50 mM PB at 220�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
S. Abbasi and S.S. Kumar
3118 The Journal of Comparative Neurology |Research in Systems Neuroscience
containing 0.5% Triton X-100, 2% goat serum (Vector,
Burlingame, CA) and 2% bovine serum albumin in 0.1 M
PB for 4 hours. 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 lg/ml) and Alexa 488 goat anti-mouse
(10 lg/ml; Molecular Probes, Eugene, OR) in blocking
solution overnight. Slices were rinsed, mounted on
slides, and coverslipped with Vectashield (Vector)
before being examined with a confocal microscope
(Leica TCS SP2 SE). Brightness, contrast, and sharp-
ness of photomicrographs were adjusted to highlight
anatomical features in Adobe Photoshop.
Sholl analysis and measurements of spinedensity
Biocytin-labeled cells were visualized through a light
microscope prior to reconstruction of soma and dendritic
arbor in Neuroleucida (MicroBrightField, Colchester, VT).
Sholl analysis of reconstructed neurons was performed in
Neuroexplorer (MicroBrightField) by placing a series of
concentric circles with increasing radii (10 lm) starting at
20 lm from the center of somata to measure total dendri-
tic length, apical and basilar dendritic length, and number
of dendritic intersection per shell. Primary dendrites,
defined as those originating directly from soma, were
counted manually from reconstructed neurons. Spine den-
sity measurements were obtained from confocal images of
apical dendrites captured with a Zeiss 363 oil-immersion
lens. Z-stacked imaged ranging between 10 and 80 lm in
depth were captured at 1-lm resolution and 1,024 3
1,024 pixels per image frame. Spine density was deter-
mined from a 100-lm-long dendritic portion in each cell
type at a fixed distance of 100 lm from the soma. We did
not discriminate spines based on any shape criteria, and
spine density refers to average number of spines per des-
ignated length of apical dendrite, where present. In Stu
cells, in which no apical dendrite could be discerned, spine
density was averaged from three of the primary dendrites
in each cell.
All statistical values are presented as mean 6 SEM.
Statistical differences were measured with the unpaired
Student’s t-test, unless otherwise indicated.
RESULTS
We recorded from 177 cells in the PrS (128 in LII
and 49 in LIII). Neurons were selected randomly under
differential interference contrast (DIC) optics, and lami-
nar location was determined visually during recordings
and confirmed through biocytin labeling and counter-
staining for NeuN immunoreactivity. Interlaminar
Figure 1. Presubicular slice preparation used in the study. A:
Low-power image of a Nissl-stained section identifying the major
anatomical landmarks: presubiculum (PrS), parasubiculum (Par),
subiculum (Sub), medial entorhinal area (MEA), angular bundle
(ab), dentate gyrus (DG), hippocampal CA1, and lateral entorhinal
area (LEA). Arrowheads indicate the mediolateral extent of LII in
PrS (region demarcated by red dashed lines). B: High-power
image of PrS highlighting LII (arrows) and various lamina (indi-
cated by roman numerals). C: Diagram of connectivity with PrS
(red arrows) of adjoining structures, including MEA, retrosplenial
cortex (RS), Sub, hippocampal CA1 and CA2, and dentate gyrus
(DG). Differences in the strength of PrS projections are indicated
by the thickness of the corresponding arrows. Scale bars 5 1
mm in A; 250 lm in B. [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
Physiology and morphology of cells in presubiculum
The Journal of Comparative Neurology | Research in Systems Neuroscience 3119
classification within PrS was according to convention
(Caballero-Bleda and Witter, 1993), with LII serving as
the cytoarchitectonically distinguishing landmark, con-
sisting of a compact layer composed mainly of densely
packed pyramidal cells and LIII comprising the less
densely packed cell layer that immediately follows LII
and extends up to the cell-sparse layer IV, a continuum
of lamina dissecans in ERC (Fig. 1A,B).
Current-clamp recordingsWhole-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 depolarization
(see Materials and Methods; Table 1). We used this infor-
mation in characterizing the different cell types prior to
classifying them using unsupervised hierarchical cluster
analysis (Fig. 2A, B). The current-clamp data were used
in formulating a classification schema for identifying the
different cell types in LII and LIII of PrS (Figs. 3, 4).
Voltage-clamp recordingsWhole-cell voltage-clamp recordings of spontaneous
(s)PSCs were obtained from neurons that were initially
current clamped to compare excitatory and inhibitory
synaptic drive to the different cell types under normal
conditions (Fig. 5, Table 2) and also to assay for
lamina-specific differences in synaptic drive to neurons
in LII and LIII (Table 3).
Cell morphologyWe assayed cell morphology in PrS using biocytin fills
of neurons that had been physiologically characterized
in an attempt to correlate morphology with physiology.
In total 118 of the recorded neurons were filled with
biocytin, from which 89 neurons with near-complete fills
TABLE 1.
Action Potential Waveform Properties of PrS Neurons1
Electrophysiological
parameter RS (n 5 114) IR (n 5 15) IB (n 5 23) Stu (n 5 7) SS (n 5 9) FA (n 5 10) LS (n 5 4)
Resting membrane potential(mV)
265 6 0.4 269 6 1 269 6 1.2 263 6 1.5 265 6 1.8 262 6 1.3 265 6 0.7
FA >> IR, IB; RS, Stu > IBSpike firing threshold (mV) 240 6 0.5 241 6 1.0 239 6 1.1 239 6 1.5 234 6 2.1 240 6 1.4 238 6 1.1
SS >> RS, IR, IB, FAEarly spike frequency
adaptation (%)40 6 1.8 47 6 5.8 75 6 2.3 n/d n/d 57 6 5.4 26 6 5.8
IB >> all; IB >> RS, IRLate spike frequency
adaptation (%)8 6 0.5 19 6 4.8 3 6 0.3 n/d n/d n/d 7 6 3.5
IR >> RS >> IBDelay (msec) 25 6 1 35 6 3 28 6 4 16 6 2 47 6 10 13 6 2 258 6 37
LS >> allAccommodative hump (mV) 2.1 6 0.2 1.3 6 0.3 7.8 6 0.9 1.8 6 0.6 n/d 3.2 6 0.9 0.1 6 0.1
IB >> all; IB >> RS, IRHalf-width (msec) 2.1 6 0.1 2.8 6 0.2 2.5 6 0.1 1.4 6 0.1 2.3 6 0.2 2.4 6 0.3 1.9 6 0.2
all > Stu; IB, IR >> RSInput resistance (MX) 328 6 7 264 6 22 349 6 26 247 6 18 274 6 19 340 6 35 141 6 15
all > LSSag ratio (%) 2.2 6 0.2 1.7 6 0.2 1.8 6 0.7 2.5 6 0.9 1.0 6 0.7 1.9 6 0.6 5.6 6 0.9
LS >> RS, IR > SS, FA, IR, IBMaximum frequency (Hz) 35 6 1.7 13 6 1.8 70 6 7.4 37 6 5.0 n/d 39 6 4.5 27 6 2.0
IB > all; IB >> RS, IRInstantaneous firing
frequency (Hz)24 6 1.0 9 6 1.0 30 6 1.8 21 6 5.0 n/d 23 6 3.2 23 6 1.5
all >> IR; IB > RS >> IRSteady-state firing
frequency (Hz)16 6 0.5 4 6 0.7 13 6 1.0 15 6 3.4 n/d n/d 17 6 1.0
all >> IR; IB,RS >> IRSD of steady-state firing
frequency (Hz)1.0 6 0.04 1.7 6 0.2 1.0 6 0.1 9.7 6 2.2 n/d n/d 1.2 6 0.1
Stu >> all; IR >> RS, IB
1Values represent mean 6 SE; n, number of cells; >, significantly greater with P < 0.05, t-test; >>, significantly greater, with P < 0.01, t-test; nd,
not determined because number of action potentials was insufficient for reliable analysis. RS, regular-spiking cell; IR, irregular-spiking cell; IB,
initially-bursting cell; Stu, stuttering cell; SS, single-spiking cell; FA, fast-adapting cell; LS, late-spiking cell. Electrophysiological parameters were
measured as described in Materials and Methods.
S. Abbasi and S.S. Kumar
3120 The Journal of Comparative Neurology |Research in Systems Neuroscience
were selected for reconstruction and quantification of
gross anatomical features (Figs. 6–8, Table 4). We
found that a majority of cells in LII had at least one api-
cal dendrite that entered LI, ultimately extending to the
pial surface (Figs. 6, 8A1). Cells in LIII, on the other
hand, typically had a single apical dendrite oriented
toward the pial surface, which in a majority of cases
reached either LII or LI (Figs. 7, 8A2). With the excep-
tion of Stu cells (Fig. 6D,H), all recorded neurons were
observed as having spine-rich dendrites (Fig. 6G,
Table 4).
Interactions between the PrS and other brain struc-
tures, especially those within parahippocampal and hip-
pocampal regions, have been described elsewhere
(Kohler, 1985; van Groen and Wyss, 1990a; Fig. 1C).
Interactions between cells in LII and LIII of PrS and
neighboring structures in this study were deduced from
observations of biocytin-filled axons of PrS cells projec-
ting to subiculum, parasubiculum, and MEA and into
the angular bundle (Figs. 6, 9, Table 4). Although mor-
phology is correlated with physiology in this study, our
classification of cell types is based exclusively on elec-
trophysiological parameters because 1) biocytin labeling
does not guarantee complete fills, 2) biocytin fills car-
ried out in acute brain slices preclude following axonal
and dendritic processes because of severing, and 3)
morphologically similar neurons might be physiologically
distinct (Fig. 8C,D, Table 4), with the potential to con-
found classification. For detailed analysis of PrS projec-
tions see Honda et al. (2008, 2011) and Honda and
Ishizuka (2004).
Cell classificationWe systematically classified cells in LII and LIII of
PrS using unsupervised hierarchal cluster analysis of
data obtained from current-clamp recordings of a total
of 177 neurons (for details see Materials and Methods).
This was achieved in two rounds. The first round
Figure 2.
Figure 2. Classification of neurons in LII and III of PrS by unsu-
pervised hierarchical cluster analysis. A: Classification based on
electrophysiological parameters (Table 1). Intersection of dendro-
gram branches, with the x-axis representing individual cells and
the y-axis representing the squared Euclidean distances between
group centroids at each branch point (longer vertical lines indi-
cate greater dissimilarity). Dashed lines indicate number of groups
as determined by the Thorndike method (see Materials and Meth-
ods). Inset: With the Thorndike method, five groups are sug-
gested in a scree plot; x-axis, clustering stages; y-axis, squared
Euclidean distance between group centroids. Individual cells are
color coded according to group membership (key for cell type
identification is shown in C). Cell types: irregular-spiking (IR),
regular-spiking (RS), initially bursting (IB), late-spiking (LS), stutter-
ing (Stu), fast-adapting (FA), single-spiking (SS). B: Classification
of cells in the multispiking group into remaining cell types (RS, IR,
IB) using cluster analysis based on physiological parameters.
C: Summary of cell classification and percentage sampled cells in
each group within PrS as a whole and in layers II and III. [Color
figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
Physiology and morphology of cells in presubiculum
The Journal of Comparative Neurology | Research in Systems Neuroscience 3121
yielded five clusters, four of which were cell types that
had been classified individually based on empirical
observations, and the last cluster of multispiking cells
(Fig. 2A) contained three cell types that displayed very
different action potential waveforms. A second round of
clustering, limited to multispiking cells, was undertaken
to refine the segregation of these cells into three differ-
ent clusters, ultimately revealing the seven distinct
types of PrS neurons addressed in our study
(Fig. 2A, B).
Regular-spiking cellsRegular-spiking (RS) cells accounted for 62% (56% in LII
and 78% in LIII) of the total cells recorded from the PrS
(Fig. 2C). RS cells were characterized by sustained fir-
ing of regularly spaced action potentials in response to
current injections or depolarization (Fig. 3A). Although
maximum firing frequency and early and late spike fre-
quency adaptation of the action potential discharge
were comparable to those of other cell types (Table 1),
RS cells had the lowest standard deviation of steady-
state firing frequency of all cell types and lacked
accommodative hump or sag altogether (Figs. 3A, 4B,
F, Table 1). The sEPSC and sIPSC frequencies and
amplitudes in RS cells were also similar to those of
other cell types (Fig. 5A, H, Table 2). RS cells had
pyramidal somata under DIC optics, with a single, spiny
apical dendrite that typically extended toward the pial
surface and ramified distally in LI (Figs. 6A,G, 7A,D, 8A,
Table 4). The basilar dendritic field of these neurons
was confined locally within the lamina of the recorded
neurons (LIII-RS cells) or adjacent lamina (LII-RS cells).
Basilar dendrites, defined as short dendritic segments
(typically <100 lm in length) emanating from somata
of neurons that otherwise have a well-identified apical
dendrite (>100 lm long), were generally oriented in a
direction opposite to the cell’s apical dendrite (Figs. 6A,
7A, 8A). Distal dendrites of some RS neurons in LIII
failed to reach the pial surface and branched instead in
LII (Fig. 9). RS cells are likely projection neurons,
because, in addition to projecting locally within PrS,
their axons tended to follow through the deep layers
extending to subiculum, parasubiculum, and MEA and
Figure 3.
Figure 3. Representative examples of intrinsic firing patterns in
the various cell types encountered in layers II and III of PrS.
Action potential waveforms of indicated cell types in response to
current injections of 100 pA (right) and step increments in mem-
brane voltage in response to 500-msec-long hyperpolarizing (250,
2100 pA) and depolarizing (100 pA) current steps (left). Resting
membrane potential of the neurons (Vm) is indicated alongside
the responses (A–G). H: Composite plot of instantaneous firing
frequency vs. time in response to depolarizing current injection of
100 pA to highlight firing frequency adaptation in the various cell
types, except for SS cells. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
S. Abbasi and S.S. Kumar
3122 The Journal of Comparative Neurology |Research in Systems Neuroscience
into the angular bundle. In a majority of cells, the axons
bifurcated to multiple brain regions (Fig. 9, Table 4).
Irregular-spiking cellsIrregular-spiking (IR) cells accounted for 8% of recorded
cells and were found exclusively in LII of PrS (Fig. 2C).
Their action potential discharge profile was character-
ized by the firing of irregularly spaced action potentials
in response to current injection or depolarization (Fig.
3B). Action potential waveforms for IR cells demon-
strated the lowest maximum firing frequency (<15 Hz),
the lowest steady-state firing frequency (<5 Hz), and
the highest late firing frequency adaptation (>15%)
compared with either RS or initially bursting (IB) cells.
IR cells had a significantly greater standard deviation of
the steady-state firing frequency (>1.5 Hz, P < 0.01;
Fig. 4, Table 1), suggesting, as their name implies, the
irregularity with which these cells fire action potentials.
sEPSC frequency of IR cells was similar to that of other
cell types, but sIPSC frequency (0.2 6 0.04 Hz) and
amplitude (34 6 2 pA) were significantly lower (P <
0.05) compared with other cells (Fig. 5B, H, Table 2). IR
cells had pyramidal somata under DIC optics, with one
or two spiny apical dendrites projecting toward the pial
surface (Figs. 6B, G, 8A1, Table 4). Basilar dendrites of
IR cells showed considerable branching that was local-
ized close to LII and was usually oriented in the direc-
tion opposite to the apical dendrites and facing toward
deep layers. IR cells also appeared to be projection
neurons, with axons extending into subiculum, deeper
layers of PrS, and the angular bundle (Table 4).
Initially bursting cellsInitially bursting (IB) cells accounted for 13% (12% in LII
and 14% in LIII) of total recorded cells (Fig. 2C) and
were characterized by the firing of an initial burst of
action potentials, followed by sustained, regularly
spaced action potentials in response to current injec-
tion or depolarization (Fig. 3C). Action potential wave-
forms for IB cells had significantly higher maximum
firing frequency (>60 Hz, P < 0.05) and early spike-
frequency adaptation (>70%, P < 0.01) and lower late
spike-frequency adaptation (<4%, P < 0.05) compared
with all other cell types (Fig. 4, Table 1). IB cells were
the only cell type to display a prominent accommoda-
tive hump (>7 mV; Figs. 3C, 4B). IB cells had moderate
sEPSC and IPSC frequencies, similar to IR cells, but sig-
nificantly smaller sIPSC amplitudes (34 6 2 mV, P <
0.05; Fig. 5C,H, Table 2). IB cells were pyramidal neu-
rons with a spiny apical dendrite that projected to the
pial surface and branched extensively in LI (Figs. 6C,G,
7B, D, 8A, Table 4). Among pyramidal cells in LII, IB
cells had the highest number of primary dendrites (5.7
6 0.4) and the greatest total basilar dendritic length
(1.1 6 0.1 mm; Table 4). Axons were routinely
observed projecting out of the PrS to MEA and/or the
angular bundle (Fig. 6C, Table 4).
Stuttering cellsStuttering (Stu) cells were found exclusively in LII and
accounted for 4% of recorded cells (Fig. 2C). Stu cells
responded to current injection or depolarization by firing
high-frequency bursts of action potentials that were inter-
mittently interrupted by periods of quiescence (Fig. 3D).
The action potential waveform for Stu cells demonstrated a
significantly higher (P < 0.05) standard deviation of the
steady-state firing frequency (>7 Hz) and smaller action
potential half-width (<1.5 msec) compared with all other
cell types (Table 1). Voltage-clamp data showed Stu cells
as having moderate sEPSC and sIPSC frequencies but
larger amplitude events relative to other cell types
(Fig. 5D, H, Table 2). Stu cells were nonpyramidal, multipo-
lar cells with an abundance of thin primary dendrites, the
largest of all cells characterized in this study (13.5 6 0.5,
P < 0.01), projecting in all directions from the cell body.
Figure 4. A–F: Summary distributions of action potential firing
properties of the indicated multispiking cell types identified dur-
ing the second round of clustering (Fig. 2B). Refer to Table 1 for
accompanying values. **P < 0.01, t-test.
Physiology and morphology of cells in presubiculum
The Journal of Comparative Neurology | Research in Systems Neuroscience 3123
Distinguishing features of Stu cells were the conspicuous
absence of an apical dendrite and very low spine density
(Figs. 6D, G, H, 8A1, Table 4). The dendritic field of Stu cells
was confined to the border between LI and LII of PrS and
did not extend into LIII. We did not observe axons from Stu
cells extending out of LII or the PrS (Table 4). This fact,
combined with the pattern of dendritic arborization and the
paucity of spines, leads us to believe that Stu cells may be
a class of local interneurons that are likely GABAergic.
Single-spiking cellsSingle-spiking (SS) cells were found exclusively in LII and
made up 5% of cells recorded from the PrS (Fig. 2C). SS
cells respond to current injection or depolarization by fir-
ing a single action potential, followed by maintained qui-
escence for the duration of the depolarization period
(Fig. 3E). SS cells displayed normal Vm, action potential
half-width, and input resistance (Table 1). Voltage-clamp
analysis indicated that excitatory and inhibitory drive to
SS cells was also similar to that of other cells (Fig. 5E,H,
Table 2). SS cells were pyramidal-like with a single, spiny
apical dendrite branching extensively in LI before reach-
ing the pial surface (Fig. 6G, Table 4). SS cells also had
a well-defined basilar dendritic field that was oriented
laterally to the cell’s apical dendrite (Figs. 6E, 8A1).
Although not quantified, apical dendrites on SS cells
appeared to be thicker than those of other cells and had
a pattern of branching that appeared more spread out in
the dorsal–ventral plane than any other cell type. We
failed to detect any axonal projections from these cells
leaving the PrS (Table 4).
Fast-adapting cellsFast-adapting cells (FA) were restricted to LII and made
up 5% of total recorded cells (Fig. 2C). FA cells respond
to current injection or cell depolarization by firing action
potentials at regularly spaced intervals for a brief period
before becoming quiescent for the remainder of the cell
depolarization. This pattern of action potential firing
was independent of current injection, because
increases in the amount of current injected did not
alter their discharge profile (Fig. 3F). Voltage-clamp
Figure 5.
Figure 5. Excitatory and inhibitory synaptic drive to PrS neurons under
normal conditions. A–G: Spontaneous excitatory postsynaptic currents
(sEPSCs; inward events recorded at 270 mV holding potential; left)
and spontaneous inhibitory postsynaptic currents (sIPSCs; outward
events recorded at 0 mV holding potential; right) measured during
voltage-clamp of the indicated cell types in layers II and III of PrS. H:
Bar plots showing mean frequencies and amplitudes of postsynaptic
currents recorded in these neurons Error bars indicate SEM. For statis-
tical comparisons see Table 1.
S. Abbasi and S.S. Kumar
3124 The Journal of Comparative Neurology |Research in Systems Neuroscience
analysis did not reveal any feature unique to FA cells,
and excitatory and inhibitory drives to these cells were
comparable to those of other cell types (Table 2, Fig.
5F,H). FA cells had a morphology similar to that of LII
RS cells with pyramidal somata that gave rise to a sin-
gle, spiny apical dendrite projecting toward the pial sur-
face and ramifying in LI. FA cells, like RS cells, also had
multiple, thin basilar dendrites that were confined to LII
(Figs. 6F, G, 8A1, Table 4).
Late-spiking cellsLate-spiking (LS) cells were the only cell type restricted
exclusively to LIII of the PrS, accounting for 8% of all cells
in that layer (Fig. 2C). LS cells were characterized by a
significantly delayed firing of action potentials following
depolarization or current injection (>200 msec), followed
by discharge of regularly spaced action potentials (Fig.
3G). The action potential waveform for LS cells was simi-
lar to that of RS cells; early spike-frequency adaptation,
late adaptation, maximum firing frequency, and standard
deviation of steady-state firing frequency were similar
between the two cell types (Table 1). In addition to being
the only cells that displayed this pattern of action poten-
tial discharge, LS cells also had a significantly lower input
resistance compared with any other cell type (<160 MX;
Table 1). Voltage-clamp analysis revealed no distinguish-
ing feature to differentiate LS cells further from the other
cells (Fig. 5G, H, Table 2). LS cells had pyramidal somata
with a spiny apical dendrite that projected toward the pial
surface (Figs. 7C,D, 8A2, Table 4). The total dendritic
length of LS cell was the greatest for all cell types in LII–
III (3.3 6 0.2 mm). Basilar dendrites tended to branch in
all directions but were confined to LIII. No axons were
observed leaving PrS in any of our labeled LS cells, sug-
gesting that these cells belonged to the nonprojecting
population of neurons within PrS (Table 4).
Laminar distinctionOur observation that RS and IB cells were common
to both LII and LIII of PrS prompted us to examine
whether there were any differences between the
TABLE 2.
Summary of Frequency and Amplitude of PSCs in PrS Neurons1
Electrophysiolgical
parameter RS (n 5 114) IR (n 5 15) IB (n 5 23) Stu (n 5 7) SS (n 5 9) FA (n 5 10) LS (n 5 4)
sEPSCsFrequency (Hz) 2.0 6 0.1 1.7 6 0.2 1.7 6 0.2 1.3 6 0.3 1.5 6 0.3 1.4 6 0.3 2.6 6 0.9
Groups not significantly different (P > 0.05)Amplitude (pA) 19 6 0.7 17 6 1.4 14 6 1.4 24 6 1.7 19 6 2.6 19 6 2.3 13 6 1.0
Stu >> IB, LS; RS > IBRMS noise (pA) 1.4 6 0.1 1.5 6 0.1 1.1 6 0.1 2.1 6 0.2 1.5 6 0.1 1.4 6 0.2 1.5 6 0.1sIPSCsFrequency (Hz) 0.6 6 0.05 0.2 6 0.04 0.5 6 0.08 0.7 6 0.14 0.5 6 0.18 0.3 6 0.08 1.0 6 0.6
Stu >> IR; RS, IB, LS > IRAmplitude (pA) 45 6 2 34 6 2 34 6 2 54 6 7 52 6 5 45 6 3 46 6 2
Stu, SS >> IR, IB; RS, FA, LS > IR, IBRMS noise (pA) 7.9 6 0.2 7.0 6 0.4 6.8 6 0.4 9.5 6 0.6 9.0 6 0.8 8.2 6 0.7 9.9 6 1.5
1Values represent mean 6 SE. Spontaneous (s) EPSC, excitatory postsynaptic current; IPSC, inhibitory postsynaptic current; RMS, root mean
square values of noise. RS, regular-spiking cell; IR, irregular-spiking cell; IB, initially-bursting cell; Stu, stuttering cell; SS, single-spiking cell; FA, fast-
adapting cell; LS, late-spiking cell. >, P < 0.05; >>, P < 0.01, t-test.
TABLE 3.
Comparison of Parameters Between Cells of LII and LIII
in PrS1
Electrophysiological parameters LII (n 5 87) LIII (n 5 45)
Voltage-clampsEPSCs
Frequency (Hz) 2.0 6 0.1 1.7 6 0.2Amplitude (pA) 17 6 1 19 6 1
sIPSCsFrequency (Hz) 0.6 6 0.1 0.6 6 0.1Amplitude (pA) 42 6 2 44 6 2
Current-clampResting pembranepotential (mV)
265 6 0.5 267 6 0.6
Early spike frequencyadaptation (%)
46 6 2.2 44 6 3.6
Late spike frequencyadaptation (%)
7 6 0.6 6 6 0.7
Delay (msec) 25 6 1.7 25 6 1.8Accommodative hump (mV) 3.2 6 0.4 2.9 6 0.5Half-width (msec) 2.3 6 0.1 1.8 6 0.1Input resistance (MX) 328 6 10 339 6 12Sag ratio (%) 2.1 6 0.2 2.2 6 0.4Maximum frequency (Hz) 40 6 2 44 6 4Instantaneous firingfrequency (Hz)
25 6 1 25 6 1
Steady-state firing frequency (Hz) 15 6 1 16 6 1SD of steady-state firingfrequency (Hz)
1 6 0.04 1 6 0.06
1Values represent mean 6 SE. None of the comparisons was statisti-
cally significant, P > 0.05, t-test.
Physiology and morphology of cells in presubiculum
The Journal of Comparative Neurology | Research in Systems Neuroscience 3125
common cell types found within the two lamina (Table
3). To test this possibility, we combined all measured
electrophysiological parameters, irrespective of cell
type (restricted to RS and IB cells), and cross-
compared these data between layers II and III. Our
results indicated no significant differences (P > 0.05)
for any of the measured variables, thereby validating
our classification of neurons based on their attributes
rather than on the laminar location of their somata
within PrS.
Sholl analysis of PrS neuronsTo characterize dendritic morphology of cell types, a
Sholl analysis from reconstructions of biocytin-filled
neurons was undertaken, followed by quantification of
total dendritic branching and length (Fig. 8B–D). We
found that the average number of intersections for Stu
cells (maximum 5 33.5 at 50 lm from soma) was sig-
nificantly greater than that of all other cell types in LII
between 20 and 90 lm (shell radius, Fig. 8C1). Simi-
larly, the number of intersections for IB cells (maximum
5 13 at 60 lm) was significantly greater than that of
RS cells (maximum value 5 8.4 at 60 lm) in LII
between 20 and 100 lm (Fig. 8C1). In LIII, the average
number of intersections for LS cells (maximum 5 18.7
at 50 lm) was significantly greater than that of RS cells
(maximum 5 9.2 at 70 lm) between 30 and 200 lm
from soma (Fig. 8C2). The differences in dendritic
length measurements were correlated with differences
in the average number of intersections for the cell
types in LII and LIII (Fig. 8D). In addition to the above-
noted differences, Sholl analysis suggests that dendritic
morphology between cell types is grossly similar,
despite distinct physiological profiles.
DISCUSSION
We obtained whole-cell recordings from 177 neurons
in LII and LIII of the PrS and by using unsupervised
hierarchical cluster analysis classified them into seven
functionally distinct cell types. We found that 1) the
PrS neuronal population to comprise RS cells (62%), IR
cells (8%), IB cells (13%), Stu cells (4%), SS cells (9%),
FA cells (5%), and LS cells (2%); 2) cell types in PrS are
differentially distributed with respect to LII and LIII,
with LII expressing all cell types except for LS cells,
and LIII expressing only RS, IB, and LS cells; 3) LII and
LIII of PrS show no significant differences when electro-
physiological parameters from cells common to both
layers are compared; 4) concomitant with functional
diversity, LII and LIII of PrS are composed of a hetero-
geneous cell population with spiny pyramidal cells and
aspiny nonpyramidal neurons. However, our quantitative
dendritic analysis suggests that pyramidal cells that are
physiologically distinct may be morphologically similar.
Furthermore, our study highlights the facts that LII and
LIII of PrS contain a neuronal population more diverse
than previously reported but consistent with the
Figure 6. Morphology of biocytin-labeled cells in LII of PrS. A–F:
Images represent typical examples of cells in each of the indi-
cated groups, acquired with a confocal microscope. Note differen-
ces in dendritic and/or axonal (triangles) arborization, pattern/
extent of ramification, and location of the cell bodies. G: High-
magnification views of dendrites of cell types in LII (A–F) indicat-
ing the presence or absence of spines. H: High-magnification
view of the boxed area in D (Stu cells). Scale bars 5 100 lm in
A (applies to A–C,E,F); 50 lm in D; 20 lm G,H.
S. Abbasi and S.S. Kumar
3126 The Journal of Comparative Neurology |Research in Systems Neuroscience
relatively large number of neurons found in this region
(Mulders et al., 1997) and that projections of the PrS
to neighboring brain structures originate from multiple
types of physiologically distinct neurons, with some
capable of influencing activity of multiple brain regions.
Cell typesWe recognize that neuronal categories can be flexible
entities; characteristics of firing patterns of cells can be
subject to change under a number of conditions, includ-
ing variations in membrane potential, somatic depolari-
zation, behavioral state of the animal, and/or in vitro
vs. in vivo recording conditions (Steriade, 2004). How-
ever, intrinsic spike characteristics of a neuron are
deemed an important determinant of its position in the
cortical circuit and may have a substantial role in deter-
mining its response properties (Agmon and Connors,
1992). Previous electrophysiological studies of the PrS
and/or subicular complex approached LII and LIII as
the combined superficial layers of the PrS (Canto et al.,
2012; Funahashi and Stewart, 1997a,b; Menendez de la
Prida et al., 2003). These studies indicated at most two
types of physiologically distinct neurons in superficial
PrS, with differences existing only between superficial
and deep layers (LV–LVI), without alluding to the physi-
ological differences between LII and LIII. A recently
published study showed all cells in LII and LIII to dis-
charge regularly in response to maintained or step cur-
rent injections and were all deemed regular-spiking
(Simonnet et al., 2013). We find that there are at least
six physiologically distinct cell types in LII, five of which
had spiny dendrites originating from either pyramidal
(RS, IR, IB, FA) or pyramidal-like (SS) neurons. The stut-
tering cells (Stu) of LII had nonpyramidal somata, had a
significantly lower spine density, and are likely GABAer-
gic (Ding and Rockland, 2001). On the other hand, LIII
had three physiologically distinct cell types (RS, IB, LS),
all of which were pyramidal, with dendrites that were
visibly spiny. It is possible that, within the population of
sampled neurons that were not filled with biocytin
(Table 4), there are additional nonstuttering types of
Figure 7. Morphology of biocytin-labeled cells in LIII of PrS. A–C: Images representing typical examples of cell types in each group
acquired with a confocal microscope. D: High-magnification views of dendrites of cell types in LIII (A–C), indicating the presence or
absence of spines. Note the dendritic features common to all cell types in this region. Scale bars 5 100 lm in A (applies to A–C); 20 lm
in D.
Physiology and morphology of cells in presubiculum
The Journal of Comparative Neurology | Research in Systems Neuroscience 3127
S. Abbasi and S.S. Kumar
3128 The Journal of Comparative Neurology |Research in Systems Neuroscience
Figure 8. Reconstruction and Sholl analysis of LII–LIII PrS neurons. A1,A2: Neurolucida reconstructions of neuron types in LII (A1) and LIII
(A2) showing laminar location of somata and dendritic morphology. B: Schematic of Sholl analysis indicating color-coded concentric shells
beginning at 20 lm from somata and incrementing radially by 20 lm. C1,C2: Quantification of the average (6SEM) number of dendritic
intersections of cell types in LII (C1) and LIII (C2) as a function of shell radius. D1,D2: Quantification of averaged total dendritic length for
the distance ranges indicated for cell types in LII (D1) and LIII (D2). ††P < 0.01 (comparisons between Stu cells and all other cell types in
LII); 11P < 0.01 (comparison between IB and RS cells in LII); **P < 0.01 (comparison between LS and RS cells in LIII). Scale bars 5
100 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
TAB
LE
4.
Gro
ssA
nato
mic
al
Ch
ara
cteri
zati
on
of
Bio
cyti
n-F
illed
Cells
inLII
–LII
Io
fP
rSin
This
Stu
dy1
RS
(LII
–LII
I)R
S(L
II)
RS
(LII
I)IR
(LII
)IB
(LII
–LII
I)IB
(LII
)IB
(LII
I)S
tu(L
II)
SS
(LII
)FA
(LII
)LS
(LII
I)
Near-
com
ple
ted
end
riti
cfills
(n5
89
)4
83
01
88
14
77
26
74
Trace
ab
leaxo
ns
28
16
12
51
05
5n/
d2
n/
d2
Untr
ace
ab
leaxo
ns
20
14
63
42
22
47
2B
ifurc
ati
ng
axo
ns
20
14
64
53
2n/
dn/
dn/
dn/
dA
xons
pro
ject
ing
out
of
PrS
21
14
74
64
2n/
dn/
dn/
dn/
dA
xons
wit
hin
PrS
22
13
94
74
3n/
d2
n/
d2
Axo
ns
pro
ject
ing
tosu
bic
ulu
m3
3n/
d3
n/
dn/
dn/
dn/
dn/
dn/
dn/
dA
xons
pro
ject
ing
toP
ar
22
n/
dn/
dn/
dn/
dn/
dn/
dn/
dn/
dn/
dA
xons
pro
ject
ing
toA
B9
63
14
22
n/
dn/
dn/
dn/
dA
xons
pro
ject
ing
toM
EA
84
4n/
d4
22
n/
dn/
dn/
dn/
dP
rim
ary
dend
rite
s(n
)4
.76
0.2
4.4
60
.25
.26
0.3
4.1
60
.55
.26
0.3
5.7
60
.44
.56
0.4
13
.56
0.5
4.6
60
.24
.56
0.5
5.7
60
.7S
tu>>
all;
IB(I
I)>
RS
(II)
,IR
,S
S;
RS
(III)>
RS
(II)
Tota
ld
end
riti
cle
ngth
(mm
)1
.96
0.1
1.8
60
.12
.06
0.2
1.8
60
.22
.26
0.3
2.1
60
.32
.56
0.5
3.1
60
.12
.26
0.2
1.9
60
.33
.36
0.2
LS>>
IR,R
S;
LS>
IB(I
I),S
S,F
A;
Stu>
IRR
SB
asi
lar
dend
riti
cle
ngth
(mm
)0
.86
0.1
0.6
60
.11
.16
0.1
0.7
60
.11
.06
0.1
1.1
60
.11
.06
0.3
3.1
60
.10
.96
0.3
0.6
60
.21
.66
0.3
Stu>>
all;
LS
,IB
(II)
,R
S(I
II)>>
RS
(II)
;LS
,R
S(I
II)>>
IR,
FA
;LS>>
RS
(III)
Ap
ical
dend
riti
cle
ngth
(mm
)1
.26
0.1
1.2
60
.11
.26
0.1
1.7
60
.21
.36
0.2
1.0
60
.21
.76
0.2
n/
d1
.36
0.2
1.3
60
.31
.66
0.4
IB(I
II)>
RS
(II)
Sp
ine
densi
ty(/
10
0l
m)
66
63
69
64
62
64
83
68
63
65
63
68
63
68
66
29
26
15
91
66
84
61
7A
ll>>
Stu
;FA
,SS>>
RS
(III);
FA>
RS
(II)
,IB
(II/
III)
1V
alu
es
ind
icate
num
ber
of
cells
ineach
gro
up
dis
pla
ying
the
ind
icate
danato
mic
alatt
rib
ute
(in
89
of
11
8b
iocy
tin
fille
dneuro
ns)
.n/
d,
Not
dete
cted
.R
S,
regula
r-sp
ikin
gce
ll;IR
,ir
regula
r-sp
ikin
gce
ll;IB
,
init
ially
-burs
ting
cell;
Stu
,st
utt
eri
ng
cell;
SS
,si
ngle
-sp
ikin
gce
ll;FA
,fa
st-a
dap
ting
cell;
LS
,la
te-s
pik
ing
cell.>
,P<
0.0
5;>>
,P<
0.0
1,
t-te
st.
Physiology and morphology of cells in presubiculum
The Journal of Comparative Neurology | Research in Systems Neuroscience 3129
GABAergic neurons, suggesting that Stu neurons (which
made up 4% of total sampled neurons; Fig. 2C) repre-
sent only a portion of all inhibitory neurons in PrS (van
Haeften et al., 1997). A definitive assessment of the
percentage of GABAergic neurons in PrS awaits further
studies using GAD in situ hybridization (Kumar and
Buckmaster, 2006). Nonetheless, the results of our
combined physiological and morphological study of the
superficial layers of PrS lead us to conclude that LII
and LIII merit consideration as functionally distinct lam-
ina. The presence of RS and IB cells in both LII and LIII
of PrS prompted us to assay for functional differences
between lamina, irrespective of cell type. By treating
these cells as one group, limited only by location of
soma, and comparing parameters between cells from
LII and cells from LIII, we were able to confirm that
neurons of a particular cell type were similar irrespec-
tive of their laminar location. We found both RS and IB
neurons in the two layers to have apical dendrites
branching in LI, suggesting that they were both likely to
receive overlapping inputs. Interestingly, sEPSC fre-
quency was greater than sIPSC frequency across cell
types in the PrS, although it is unclear whether these
differences are attributable to variations in signal-to-
noise at the two holding potentials or to differences in
the excitatory and inhibitory synaptic drives to the neu-
rons. Nonetheless, the synaptic drive to neurons in LII
was comparable to that in LIII, despite interneuronal
variability within the two layers. Taken together, these
findings reconcile our consideration of PrS neurons as
members of a particular cell group irrespective of their
laminar location.
PrS projection neuronsThe PrS and its surrounding regions (subiculum, para-
subiculum) have been the focus of several anatomical
tracing studies (Caballero-Bleda and Witter, 1993;
Honda and Ishizuka, 2004; Kohler, 1985; Kononenko
and Witter, 2012). These studies all indicate a promi-
nent ipsilateral projection from the PrS to LIII of MEA
that originates mostly in LIII of PrS and to lesser extent
in LII. Our study showed axons originating from PrS
cells whose somata were in LII and LIII projecting to
subiculum, parasubiculum, angular bundle, and LIII of
MEA. Two of the cell types, the RS and IB cells, had
axons that extended beyond the deep layers of PrS into
MEA via LV–VI, ultimately branching in LIII of MEA, the
potential location of neurons targeted by the PrS
(Caballero-Bleda and Witter, 1994; van Haeften et al.,
Figure 9. Confocally acquired montage of a bifurcating axonal projection from a biocytin-labeled RS cell in LIII of PrS. Note the clean bifur-
cation of the axon (arrowheads) in LV–VI of PrS and its projection into LIII of MEA and into the angular bundle (ab). Inset: High-
magnification view of the point of axonal bifurcation. Dashed lines indicate boundaries of parahippocampal structures in relation to the
PrS and the pial surface (solid line). Scale bars 5 100 lm; 20 lm in inset.
S. Abbasi and S.S. Kumar
3130 The Journal of Comparative Neurology |Research in Systems Neuroscience
1997; Wouterlood et al., 2004). Principal neurons in LIII
of MEA are highly vulnerable in TLE, with severe cell
loss observed in both patients (Du et al., 1993) and ani-
mal models of the disease (Du et al., 1995). It is postu-
lated that the synaptic input from PrS plays a
prominent role in this pathology in part because of the
sparing of neurons in neighboring lateral entorhinal area
(LEA) that lacks a prominent PrS input (Eid et al., 1996,
2001). Furthermore, this projection has proved to be of
interest recently in the control of neural networks that
underlie maintenance of an animal’s navigational abil-
ities mediated via the retrosplenial–PrS–MEA axis
(Kononenko and Witter, 2012).
Another aspect worth considering in the context of
afferent projections provided by PrS to the MEA is
bidirectionality of information transfer; we observed
that axons originating from at least two neuronal types
(RS and IB cells) could bifurcate to LIII of MEA as well
as to the angular bundle (Fig. 9). The locus of these
axonal bifurcations appeared to be independent of
somatic location and was generally confined to deep
PrS, at the level of the angular bundle. In addition to
coupling MEA and the angular bundle, we observed
neurons (IR and RS cells) whose bifurcating axons also
coupled activities of deep PrS and subiculum or para-
subiculum (Table 4). Recently, bidirectional axonal col-
laterals were described for LV–VI of PrS (Honda et al.,
2011) and were proposed to mediate synchronization
of coordinated neuronal firing between spatially dis-
tributed regions aiding hippocampal–parahippocampal
memory circuits. Bidirectional axons originating from
cells of PrS projecting to MEA could serve similar roles
in circuits that involve LIII of MEA, the temporoam-
monic pathway, and CA1 of the hippocampus.
Although the afferents that PrS provides to LIII of MEA
have been well documented, the physiological profiles
of neurons that give rise to these projections are
much less well understood. Our study pinpoints the
identity and location of these projection neurons
within the PrS. Compared with the hippocampus and/
or ERC, the PrS is a relatively obscure area whose
exploration is likely to aid in a better understanding of
the interactions between various structures of the
temporal lobe.
ACKNOWLEDGMENTSThe authors thank Dr. Jyotsna Pilli for helpful discus-
sions throughout the course of this study, Max Richardson
for assistance with immunohistochemistry, and Ruth Did-
ier for assistance with confocal microscopy. We are also
grateful to Mark Basista, Deirdre M McCarthy, Molly
Foote, Dr. Bhide, and Dr. Johnson and for their guidance in
neuronal reconstructions and microscopy.
CONFLICT OF INTEREST STATEMENT
The authors have no conflicts of interest to declare
ROLES OF AUTHORS
Conception and design of research: SA, SSK; per-
formed experiments: SA, SSK; analyzed data: SA, SSK;
interpreted results of experiments: SA, SSK; approved
final version of manuscript: SA, SSK; prepared figures:
SA, SSK; drafted manuscript: SA; edited and revised
manuscript: SSK.
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