electrophysiological and morphological characterization of cells in superficial layers of rat...

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: [email protected]

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.]



wileyonlinelibrary.com

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.



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.]




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.]




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.



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.



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.



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.



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.





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.



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.



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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