neurogliaform cells in the molecular layer of the dentate gyrus as feed-forward γ-aminobutyric...

Neurogliaform Cells in the Molecular Layer of theDentate Gyrus as Feed-Forward c-AminobutyricAcidergic Modulators of Entorhinal–HippocampalInterplay

Caren Armstrong,1* Janos Szabadics,1 Gabor Tamas,2 and Ivan Soltesz1

1Department of Anatomy and Neurobiology, University of California, Irvine, School of Medicine, Irvine, California 926972HAS Research Group for Cortical Microcircuits, Department of Comparative Physiology, University of Szeged,

H-6726 Szeged, Hungary

ABSTRACTFeed-forward inhibition from molecular layer interneur-

ons onto granule cells (GCs) in the dentate gyrus is

thought to have major effects regulating entorhinal–hip-

pocampal interactions, but the precise identity, proper-

ties, and functional connectivity of the GABAergic cells in

the molecular layer are not well understood. We used sin-

gle and paired intracellular patch clamp recordings from

post-hoc-identified cells in acute rat hippocampal slices

and identified a subpopulation of molecular layer inter-

neurons that expressed immunocytochemical markers

present in members of the neurogliaform cell (NGFC)

class. Single NGFCs displayed small dendritic trees, and

their characteristically dense axonal arborizations covered

significant portions of the outer and middle one-thirds of

the molecular layer, with frequent axonal projections

across the fissure into the CA1 and subicular regions.

Typical NGFCs exhibited a late firing pattern with a ramp

in membrane potential prior to firing action potentials,

and single spikes in NGFCs evoked biphasic, prolonged

GABAA and GABAB postsynaptic responses in GCs. In

addition to providing dendritic GABAergic inputs to GCs,

NGFCs also formed chemical synapses and gap junctions

with various molecular layer interneurons, including other

NGFCs. NGFCs received low-frequency spontaneous syn-

aptic events, and stimulation of perforant path fibers

revealed direct, facilitating synaptic inputs from the ento-

rhinal cortex. Taken together, these results indicate that

NGFCs form an integral part of the local molecular layer

microcircuitry generating feed-forward inhibition and pro-

vide a direct GABAergic pathway linking the dentate gyrus

to the CA1 and subicular regions through the hippocam-

pal fissure. J. Comp. Neurol. 519:1476–1491, 2011.

VC 2010 Wiley-Liss, Inc.

INDEXING TERMS: hippocampus; GABA; gap junction; perforant path; interneuron

The dentate gyrus is a region uniquely situated to

control the effects of incoming cortical inputs on the

hippocampus. The perforant path, formed by cells of

layer II in the entorhinal cortex, constitutes the main

input to the dentate gyrus (Steward, 1976; Varga

et al., 2010). Although significant excitatory input

arrives at the dentate through the perforant path, few

granule cells (GCs) will fire to pass along this input to

the CA3 region. This is due to the relatively low excit-

ability of GCs, which is subserved both by the intrinsic

properties of the GCs and also by the local GABAergic

microcircuits within the dentate (Fricke and Prince,

1984; Mody et al., 1992a,b; Scharfman, 1992; Staley

et al., 1992; Williamson et al., 1993; Coulter, 1999;

Nusser and Mody, 2002; Pathak et al., 2007). The nor-

mal entorhinal–hippocampal circuit is involved in per-

forming pattern separation and in spatial information

encoding (Leutgeb et al., 2007; Moser et al., 2008),

and, for both of these functions, the sparse firing of

GCs is important.

Additional Supporting Information may be found in the online version ofthis article.

J. Szabadics’s current address is Institute of Experimental Medicine,Hungarian Academy of Sciences, Szigony utca 43, Budapest 1083,Hungary.

Grant sponsor: National Institutes of Health; Grant number: NS35915;Grant sponsors: UC Irvine medical scientist training program (to C.A.);George E. Hewitt Foundation for Medical Research (to J.S.).

*CORRESPONDENCE TO: Caren Armstrong, Department of Anatomy andNeurobiology, Irvine Hall Rm. 139, University of California, Irvine, Irvine,CA 92697-1280. E-mail: [email protected]

VC 2010 Wiley-Liss, Inc.

Received August 24, 2010; Revised October 16, 2010; AcceptedDecember 10, 2010

DOI 10.1002/cne.22577

Published online December 23, 2010 in Wiley Online Library(wileyonlinelibrary.com)

1476 The Journal of Comparative Neurology | Research in Systems Neuroscience 519:1476–1491 (2011)

RESEARCH ARTICLE

Furthermore, the low excitability of dentate GCs has

led to the region being referred to as the ‘‘dentate gate,’’

preventing overexcitation of the CA regions of the hippo-

campus under normal conditions (Andersen et al., 1966;

Heinemann et al., 1992; Lothman et al., 1992; Behr et al.,

1998; Hsu, 2007; Pathak et al., 2007) and possibly play-

ing an important role in pathological conditions such as

epilepsy, in which the gate function can transiently break

down, allowing excessive excitation to reach the recur-

rent network of the CA regions (Heinemann et al., 1992;

Lothman et al., 1992; Mody et al., 1992b; Sloviter and

Brisman, 1995; Behr et al., 1998; Coulter, 1999; Ang

et al., 2006; Hsu, 2007; Pathak et al., 2007). Thus, under-

standing the normal function of the dentate is crucial to

understanding pathological conditions.

In the hilus and granule cell layers of the dentate gyrus,

the microcircuits involving GABAergic interneurons and

GCs have been explored, and both feed-forward and feed-

back interneurons onto GCs have been described

(Buzsaki, 1984; Seress and Ribak, 1984; Sloviter, 1991;

Freund and Buzsaki, 1996; Penttonen et al., 1997;

Kraushaar and Jonas, 2000; Alle et al., 2001). However,

although careful descriptions of individual molecular layer

interneurons exist (Seress and Ribak, 1983; Soriano and

Frotscher, 1989; Halasy and Somogyi, 1993; Han et al.,

1993; Freund and Buzsaki, 1996; Chittajallu et al., 2007;

Capogna, 2011), the precise identities of interneurons

performing feed-forward roles in this layer have yet to be

delineated. Because of the anatomical proximity of mo-

lecular layer interneurons to incoming perforant path

input, feed-forward interneurons in the molecular layer

would be expected to contribute to the sparse firing

observed in dentate GCs. This role may be crucial in

proper circuit function. Indeed, feed-forward inhibition

has recently been shown to have dramatic computational

effects on network dynamics (Ferrante et al., 2009).

For the molecular layer, we found that a subpopulation

of interneurons was positive for markers matching the pro-

file of cells belonging to the neurogliaform cell (NGFC) fam-

ily, comprising NGFCs [originally described by Cajal as

arachniform cells (Ramon y Cajal, 1999)] and ivy cells,

which possess unique properties that have recently been

described for the neocortex and CA3 and CA1 regions (Del-

ler and Leranth, 1990; Vida et al., 1998; Tamas et al.,

2003; Price et al., 2005, 2008; Simon et al., 2005; Houser,

2007; Olah et al., 2007; Szabadics et al., 2007, 2010; Elfant

et al., 2008; Szabadics and Soltesz, 2009; Karayannis

et al., 2010). Interneurons of the NGFC family release

GABA from many sites into the extracellular space and can

activate even extrasynaptic GABA receptors with a single

action potential. This has led to the suggestion that cells of

the NGFC family exhibit a unique type of neurotransmission

separate from synaptic or electrical coupling—volume trans-

mission (Olah et al., 2009; Capogna & Pearce, 2011).

NGFCs induce both a slow GABAA-mediated as well as

delayed and prolonged GABAB-mediated current, resulting

in a large charge transfer upon stimulation (Tamas et al.,

2003; Szabadics et al., 2007). Such properties enable

these cells to mediate slow but long-lasting, powerful con-

trol of their targets. Additionally, whereas most interneur-

ons form electrical synapses only with other cells of the

same type, NGFCs are known to form promiscuous gap

junctions with other interneuronal types, perhaps enabling

them to coordinate the activity of diverse players in local in-

hibitory microcircuits (Simon et al., 2005; Zsiros and Mac-

caferri, 2005, 2008; Olah et al., 2007). A feed-forward inter-

neuron with the unique features of the NGFC in the

molecular layer would be well-suited to play a role in main-

taining strict control of GC excitability.

The diversity of molecular layer interneurons, particu-

larly insofar as these cells may correspond to known cell

types, has not been fully explored, and here we identify

and characterize NGFCs as a specific interneuronal sub-

type present in the dentate molecular layer. Defining

such specific microcircuitry in this region will aid in under-

standing the normal dentate and how and why it may

change in pathological conditions.

MATERIALS AND METHODS

AnimalsMale and female adolescent (3–5 weeks postnatal)

Wistar rats (Charles River, Wilmington, MA) were deeply

anesthetized with isofluorane for acute hippocampal slice

electrophysiology and subsequent immunocytochemistry.

All experimental protocols involving animals were

reviewed and approved by the UC Irvine Institutional Ani-

mal Care and Use Committee.

ElectrophysiologyCells were recorded from acute horizontal hippocam-

pal slices (350 lm) from the ventral hippocampal forma-

tion prepared in ice-cold sucrose solution (containing in

mM: 85 NaCl, 75 sucrose, 2.5 KCl, 25 glucose, 1.25

NaH2PO4, 4 MgCl2, 0.5 CaCl2, 24 NaHCO3), incubated for

1 hour at 32�C, and stored at room temperature until re-

cording in ACSF (containing in mM: 126 NaCl, 2.5 KCl, 26

NaHCO3, 2 CaCl2, 2 MgCl2 1.25 NaH2PO4, 10 glucose).

Slices were visualized using an Eclipse FN-1 (Nikon)

microscope with infrared (750 nm) Nomarski differential

interference contrast optics (Nikon 40XNIR Apo N2

NA0.8W WD3.5 objective with �1.5 magnification lens)

and recorded at 36�C 6 0.5�C. Unless otherwise speci-

fied, whole-cell somatic recordings were made using 3–5

MX borosilicate glass pipettes filled with intracellular so-

lution containing (in mM): 90 K-gluconate, 1.8 NaCl, 1.7

Dentate gyrus neurogliaform cells

The Journal of Comparative Neurology | Research in Systems Neuroscience 1477

MgCl2, 27.4 KCl, 0.05 EGTA, 10 HEPES, 2 MgATP, 0.4

Na2GTP, 10 phosphocreatine, and 8 biocytin (pH 7.25,

270–290 mOsm). Recordings were made and controlled

using a MultiClamp 700B amplifier (Molecular Devices,

Union City, CA), and Clampex software (version 9.2; Axon

Instruments, Burlingame, CA).

Drug applicationDrugs were obtained from Tocris (Ellisville, MO), dis-

solved in ACSF, and bath applied at the following concen-

trations: 10 lM D-APV, 5 lM NBQX, 50 nM CGP55845, 5

lM gabazine (SR95531).

Intrinsic propertiesRecordings were used only from cells originally

patched and recorded in normal ACSF and confirmed as

NGFCs by both firing pattern and axonal morphology (see

below). Input resistance was calculated from the steady-

state voltage change induced by the last 300 msec of a

small (�20 pA) 1-second hyperpolarizing current step. Sag

was measured by comparing the negative voltage peak

during the first 100 msec of a�100 pA 1-second hyperpo-

larizing pulse with the steady-state voltage during the last

300 msec of the same pulse. Membrane time constant

was measured from the best fit single exponential curve of

the voltage response to the beginning of a �100-pA step.

Action potential properties were determined for the first

three spikes elicited in a cell and averaged.

Paired recordingsPresynaptic interneurons were held near �60 mV in

current clamp. One or two short [2-msec duration; 2.5-

msec interstimulus interval (ISI)] current pulses were

delivered to evoke single or dual action potentials. Postsy-

naptic cells were held in voltage clamp. To establish

whole-cell recordings for pairs, an extracellular solution

with low Ca2þ concentration (0.15 mM) was typically used

to patch the NGFC to avoid overexcitation during patch

formation (Tamas et al., 2003); once the preparation was

stable, whole-cell recording was achieved in the NGFC

and the presumed postsynaptic cell, and the perfusate

was switched to ACSF containing normal extracellular

[Ca2þ] to test for connections between the cells. Sweeps

from individual experiments were averaged, and analyses

of kinetics were performed from averaged traces. Ampli-

tude, 10–90% rise time, and decay time constant were

measured for GABAA at �90 mV, where GABAB is close to

its reversal potential. For GABAB kinetics, current

response in the GABAB antagonist CGP55845 was normal-

ized by the GABAA peak in normal ACSF with the postsy-

naptic cell at �50 mV and subtracted to give an approxi-

mation of the GABAB component alone. Additionally, a

measure of the magnitude of the GABAB response was

given by the area under the GABAB portion of the curve.

The current recorded in whole-cell voltage clamp mode in

the postsynaptic cell was expressed as a percentage of

the hyperpolarizing current injected into the presynaptic

cell to generate the average coupling coefficient of electri-

cally coupled interneurons.

Field stimulation and spontaneouspostsynaptic current measurements

Constant-current stimuli (10 lsec) were applied at

0.1 Hz through a bipolar 90-lm tungsten stimulating elec-

trode placed in the subiculum within 100 lm of the hippo-

campal fissure. A stimulus isolator (WPI A360D) was used

to gradually increase the stimulus strength until a stable

response could be observed. To ensure that only monosy-

naptic responses were considered, data were rejected if

the latency between stimulation and response was

greater than 3 msec. Paired pulses were delivered at ISIs

of 50, 100, 150, or 200 msec. Once a stable amplitude

inward current had been established in control con-

ditions, evoked responses were recorded in ACSF with

sequential and additive washes in of gabazine and

CGP55845 together, D-APV, and finally NBQX; in each

case, at least 15 sweeps after wash-in were averaged,

and changes in current amplitude were normalized to am-

plitude in normal ACSF. In a subset of field stimulation

experiments, NBQX was washed out until the inward cur-

rent reappeared (10–20 minutes). The responses elicited

by paired perforant path stimulations were normalized to

the amplitude of the first peak, and paired-pulse ratio

was expressed as a ratio of second:first peak amplitude.

Spontaneous postsynaptic current (sPSC) frequencies

in NGFCs and GCs were determined from 1-minute-long

data segments in normal ACSF. Analysis of sPSCs was

performed using the MiniAnalysis program (version 6.0.7;

Synatptosoft) with visual inspection of detected events.

For sIPSC analysis, cells were patched using a CsCl-

based internal solution (containing, in mM: 40 CsCl, 90

K-gluconate, 1.8 NaCl, 1.7 MgCl2, 3.5 KCl, 0.05 EGTA, 10

HEPES, 2 MgATP, 0.4 Na2GTP, 10 phosphocreatine, 8 bio-

cytin, pH 7.2, 270–290 mOsm) in ACSF containing D-APV

and NBQX.

Cell identificationNGFCs were positively identified based on firing prop-

erties, synaptic characteristics, and axonal morphologies.

In cases of individual NGFCs, a late-spiking firing pattern

with hyperpolarized (near �75 mV) RMP, a relatively fast

time constant, and little to no sag during hyperpolarizing

pulses were suggestive of NGFC identity; however, all

neurons were recovered with diaminobenzidine (DAB)

staining (see below), and the axon was inspected for the

Armstrong et al.

1478 The Journal of Comparative Neurology |Research in Systems Neuroscience

characteristic dense arborization and frequent en pas-

sant boutons. Cells were discarded if the axon could not

be inspected. In the case of paired recordings between

NGFCs and GCs, NGFCs were included without DAB

axon recovery only if they elicited a GABAB current in

the postsynaptic cell with a single presynaptic spike,

because this property is unique to cells of the NGFC

family (Tamas et al., 2003). GCs were identified by their

distinct firing pattern and location in the granule cell

layer. Parvalbumin (PV)-expressing basket cells (PVBCs)

were identified by their fast spiking firing pattern, PV

immunocytochemistry, and basket cell axonal morphol-

ogy in the granule cell layer by DAB.

Immunocytochemistry and morphologySlices were immediately fixed postrecording in 0.1 M

phosphate buffer (PB; pH 7.4) containing 4% paraformal-

dehyde and 0.1% picric acid for 24–48 hours at 4�C and

were resectioned at 60 or 100 lm. For 60-lm sections,

primary antibodies were used at 1:1,000 concentration:

PV (polyclonal rabbit antibody; Swant, Bellinzona, Switzer-

land), neuropeptide Y (NPY; polyclonal rabbit antibody;

Bachem, Torrance, CA), neuronal nitric oxide synthase

(nNOS; polyclonal rabbit antibody; Cayman, Ann Arbor,

MI), COUP TFII (chicken ovalbumin upstream promoter

transcription factor 2; monoclonal anti-human mouse

antibody clone H7147; Invitrogen, Carlsbad, CA), and

reelin (monoclonal a.a. 164–496 mouse antibody clone

G10; Millipore, Bedford, MA). Slices were incubated over-

night in TBS buffer (pH 7.4) containing 0.25% Triton X-

100 and 2% normal goat serum. Immunoreactions were

revealed using Alexa-488 or Alexa-594-conjugated sec-

ondary goat antibodies against rabbit or mouse, and bio-

cytin was revealed using Alexa-350-conjugated streptavi-

din. All sections were processed (with or without

immunocytochemistry) to reveal the fine details of mor-

phology using a conventional DAB staining method.

Briefly, endogenous peroxidase activity was blocked with

1% H2O2, and slices were incubated with ABC (avidin-bio-

tin complex) reagent (Vectastain ABC kit; Vector Labora-

tories, Burlingame, CA) in 0.1% Triton X-100. The reaction

was developed with DAB and NiCl2 for 8–15 minutes and

stopped with H2O2 solution. Sections were dehydrated

and mounted. Cells were visualized with conventional

transmitted light microscopy (Zeiss Axioskop 2). Camera

Lucida drawings were made from either a single, repre-

sentative 100-lm section or reconstructed from serial

60-lm sections using a �100 oil immersion objective.

Interbouton distances were measured by light micros-

copy in six different fields of view (each 87 � 65 lm) per

cell from seven different confirmed NGFCs.

Statistical analysisAverage values are expressed as mean 6 SEM. All

wash-in experiments and paired-pulse amplitudes were

compared by two-tailed paired t-tests. All experiments

comparing groups of different cells were compared by

two-tailed unpaired t-tests.

Antibody characterizationFor all antibodies used in this study, the pattern of

expression in hippocampal slices was compared with

previously published results as cited in Table 1 and

detailed below and was consistent with these expres-

sion patterns.

Specificity dataCoup-TFII

The antibody was tested in a knockout (KO) mouse (Qin

et al., 2007); in this paper, embryonic, P0, P7, and P21 cer-

ebellum sections of COUP-TFII conditional knockout mice

[generated by Cre recombination of a COUP-TFII gene

flanked by Flox sites (F/F), with Cre expression driven by

the neuron-specific enolase (NSE) promoter] were com-

pared with the null mutant, and knockout (KO) animals did

not express COUP-TFII. Additionally, the expression pat-

tern in hippocampus was similar to published hippocampal

expression patterns (Fuentealba et al., 2010).

nNOSThe antibody was tested in a KO mouse with targeted

disruption of nNOS exon 2 in heart tissue, where no stain-

ing was observed in the KO (Dawson et al., 2005). Label-

ing pattern in the hippocampus was also similar to that

seen with other nNOS antibodies (Fuentealba et al.,

2008; Tricoire et al., 2010).

TABLE 1.

Antibody Characterization

Name Structure of immunogen Manufacturer and catalog No. Species Clonality

COUP TFII Human COUP TFII amino acids 43–64 Invitrogen; PP-clone H7147-00 Mouse MonoclonalnNOS Recombinant human nNOS amino acids 1422–1433 Cayman; 160870 Rabbit PolyclonalNPY Antigen sequence: H-YPSKPDNPGEDAPA Bachem; T-4070.0050 Rabbit Polyclonal

EDMARYYSAKRHY INLITRQRY-NH2

PV Rat muscle PV Swant; PV-28 Rabbit Polyclonalreelin Recombinant reelin amino acids 164–496 Millipore; AB5364 clone G10 Mouse Monoclonal



NPYThe antibody was tested by the manufacturer and in

addition was tested in a tetracycline-driven conditional

KO mouse in which levels of mRNA and protein were

reduced to <15% of control animals’ levels of mRNA and

protein after 9 weeks of doxycycline treatment, and the

antibody staining of the brain was comparably reduced at

that time point (Ste. Marie et al., 2005). The expression

pattern of NPY was also similar to published literature on

NPY expression in the hippocampus (Deller and Leranth,

1990; Karagiannis et al., 2009; Tricoire et al., 2010).

PVThe antibody was tested by Western blot in normal and

KO mice by using tissue from three different muscle

groups (panniculus carnosus, extensor digitorum longus,

and abdominal muscles), revealing a single band repre-

senting PV on Western blot with this antibody (Schwaller

et al., 1999). Additionally, the expression pattern matched

published data on PV-expressing neurons in the hippocam-

pus (Kosaka et al., 1987).

ReelinThis antibody was tested by ELISA and Western blot

against different epitopes of the reelin protein. This anti-

body recognized the H epitope near the N terminus of the

protein (de Bergeyck et al., 1998). Additionally, it was

tested and showed no staining in the reeler mouse, a

mouse with a large mutation within the reelin protein, and

was also tested in the Orleans reeler mouse, which has a

different C terminal frameshift mutation but a normal N

terminus, in which staining was present (de Bergeyck

et al., 1997). Furthermore, the expression pattern of this

antibody was consistent with published hippocampal

expression patterns of reelin (Pesold et al., 1998).

RESULTS

Whole-cell patch clamp recordings of NGFCs were

made in 57 acute slices from 42 animals, yielding a total

of 60 confirmed recorded NGFCs in the molecular layer

of the dentate gyrus to be used for this study. During re-

cording, putative NGFCs were identified by firing pattern

and filled with biocytin for post hoc identification. All 60

cells were confirmed either by axonal morphology or, for

a subset (n ¼ 2) of NGFC–GC pairs, by ability to elicit a

GABAB response with a single action potential (see Mate-

rials and Methods).

Single-cell characteristics of dentategyrus NGFCs

Dentate NGFCs had a typical, very dense arborization

that could be appreciated in 100-lm representative sec-

tions of recorded slices (Fig. 1A; location of cell in hori-

zontal slice shown in Fig. 1B) with frequent (average inter-

bouton distance 2.3 6 0.08 lm; n ¼ 1,930 interbouton

intervals from 42 fields of view from seven cells) en pas-

sant boutons (Fig. 1C). Filled NGFCs in the dentate con-

tained a number of markers typical of NGFCs in other

brain areas, including COUP TFII (n ¼ 16 of 19 tested

cells; Fig. 1D,E), nNOS (n ¼ 12 of 16 tested cells;

Fig. 1F,G), reelin (n ¼ 10 of 10 tested cells; Fig. 1F,H),

and NPY (n ¼ 9 of 31 tested cells; Fig. 1I,J). Note that

these numbers likely underestimate the true presence of

cellular markers in this population, because, although

a positive immunoreaction is conclusive, intracellular re-

cording allows both dilution of intracellular markers by

pipette solution and leakage of cellular contents from the

disrupted membrane after the withdrawal of the pipette,

which can lead to a false negative, particularly for weakly

staining cytoplasmic markers such as NPY.

Among 11 NGFCs in which the origin of the axon within

the dense axonal cloud could be determined with particu-

lar clarity, five originated from a dendrite and six origi-

nated from the cell body. The axon of dentate NGFCs typi-

cally spanned hundreds of micrometers along the outer

border of the dentate gyrus, primarily in the middle and

outer molecular layers but occasionally passing into the

inner molecular layer. In 11 of 17 cells that had axons

and dendrites that could be especially well visualized af-

ter DAB processing, the axon, but not the dendrite,

extended into the subicular and CA1 regions (Fig. 2A–C).

In contrast, GC dendrites never extended across the hip-

pocampal fissure to the subiculum or to the CA1 region.

To summarize the location of NGFCs and the extent of

their axons and dendrites in horizontal sections, the same

17 cells were examined under the light microscope and

their axonal and dendritic arborizations plotted onto a

flattened representation of the layers of the dentate gyrus

molecular layer (Fig. 2A,B). Axonal arbors of NGFCs on av-

erage covered 37.7% 6 1.8% of the total length (1,122.9

6 62.4 lm) of the molecular layer in horizontal sections,

as measured from inner to outer leaflet parallel to the

granule cell layer. Cell bodies of NGFCs were located in

middle (n ¼ 8) and outer (n ¼ 9) molecular layers. Com-

pared with the extensive axonal cloud, dendritic fields

were small, extending only tens of micrometers from the

cell body within the middle and outer molecular layers,

only occasionally extending into the inner molecular layer

and never extending into the granule cell layer or across

the hippocampal fissure.

Intrinsic properties were analyzed in a subset of

recorded neurons (n ¼ 21) that originally had been

patched and recorded in normal ACSF solution and had a

stable resting membrane potential in current clamp with

no current injection. Intrinsic properties were comparable

Armstrong et al.


to those seen in other brain areas and species (Povysheva

et al., 2007; Tricoire et al., 2010). NGFCs had a charac-

teristically late firing pattern, often exhibiting a ramp in

membrane potential prior to firing action potentials

(Fig. 3A, top trace), a resting membrane potential of

�75.6 6 0.9 mV, a threshold of �30 6 1 mV, an input

resistance of 147 6 8 MX, a membrane time constant

of 6.81 6 0.36 msec, and essentially no sag (0.17 6

0.10 mV) in response to a �100 pA hyperpolarizing step.

Because there was no sag, the measurement of the mem-

brane time constant in response to a �100 pA pulse was

not complicated by this parameter.

NGFC output to GCsAmong 59 paired recordings of molecular layer inter-

neurons with late-spiking firing patterns and GCs, 32

were connected (54%) and 14 connected pairs were con-

firmed as NGFCs (see Materials and Methods); 11 of the

14 connected NGFC pairs could be observed with a single

presynaptic action potential. No GC to NGFC connections

were observed in any of the 59 paired recordings.

Paired recordings between NGFCs in the molecular

layer and GCs demonstrated characteristic biphasic post-

synaptic responses consisting of two components, an

early GABAA mediated response and a late-appearing pro-

longed GABAB component (Fig. 3B; n ¼ 9 used for analy-

sis, n ¼ 2 identified only by GABAB component). These

two components could be separated in two ways, by

reversal potential and pharmacologically. The early iono-

tropic GABAA component was outward at �50 mV and

inward at �90 mV (Fig. 3B,C). The GABAB component,

mediated by a metabotropic and therefore slower Kþ cur-

rent, appeared as a late prolonged outward current at

�50 mV but not at �90 mV, close to its reversal poten-

tial. This late GABAB component, but not the early GABAAcomponent, could also be blocked by the specific GABABantagonist CGP55845 (Fig. 3C–E; n¼ 4).

The late component of the postsynaptic current

response was examined with the postsynaptic GC held in

voltage clamp at �50 mV. At this potential, both the

GABAA and GABAB currents were outward in nature.

Because single action potentials in dentate NGFCs gener-

ated very-small-amplitude responses in GCs, we used two

action potentials in quick succession (2.5-msec ISI) to

generate larger responses that could be examined more

easily for quantification of the effects of CGP. The

increase in GABAA amplitude and the area of the GABABcomponent were compared by the ratio of the response

to two action potentials vs. one action potential (2AP:1AP

ratio) in NGFC to GC pairs. The 2AP:1AP ratio for GABAAcurrent amplitude was 2.4 6 0.5 (n ¼ 7; P ¼ 0.03) and

for the area under the GABAB current was 2.6 6 0.5 (n ¼7; P < 0.01), demonstrating that both components ex-

hibit significant facilitation with 2APs. This facilitation

allowed us to investigate the nature of the late compo-

nent of the biphasic response more easily. To confirm

that the GABAB receptor was responsible for the late

component, two action potentials were used to elicit a

Figure 1. Characteristic morphologies and immunocytochemical profiles of NGFCs in the dentate. A: Camera lucida drawing from a represen-

tative 100-lm section of a dentate NGFC. B: Position of the cell shown in A within the horizontal hippocampal section. ML, molecular layer;

GCL, granule cell layer. C: Light microscopic view (�100) of the characteristic axonal arborization of the cell shown in A showing multiple axo-

nal branches passing through a single plane of focus and frequent, small en passant boutons. D–J: Recorded and confirmed NGFCs in the den-

tate filled with biocytin (�100) express a variety of NGFC markers, including COUP TFII (D,E), nNOS (F,G), and reelin (G), and NPY (H–J). Each

image represents a single fluorescent channel, with biocytin indicating the recorded cell (D,F,I) and the adjacent panels indicating individual

antibody labeling in the same plane of focus (E,G,H,J). The brightness and contrast of these images were digitally adjusted to provide maximal

visualization of the markers. Scale bars ¼ 20 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]



large response, and the area under the GABAB curve from

the end of the GABAA response to the end of the GABABresponse was compared before and after wash-in of the

GABAB antagonist CGP55845. In these experiments, the

late component was abolished by CGP55845 (n ¼ 4; late

component area in ACSF 1,143.6 6 178.4 pAms, in

CGP55845 49.5 6 89.7 pAms; P ¼ 0.02), demonstrating

that the late component was, indeed, mediated by GABABreceptors. In a subset of cases in which a clear GABABresponse was elicited with a single action potential (Fig.

3B; n ¼ 3), CGP55845 was used to estimate the kinetics

of the GABAB current. To do so, the remaining average

GABAA response to a single NGFC action potential in the

presence of CGP55845 was subtracted from the average

biphasic GABAA- and GABAB-mediated response in nor-

mal ACSF to generate an estimate of unitary GABAB cur-

rent alone (10–90% rise time 71.5 6 6.8 msec, decay

from peak to 50% amplitude 94.3 6 8.6 msec, amplitude

8.99 6 1.14 pA, area under GABAB curve 963.1 6

241.5 pAms). Note that, for CGP55845, the kinetics of

the GABAA responses to 1AP were not significantly

changed between control and CGP as measured at �90

mV (n ¼ 3; 10–90% rise time in ACSF 4.2 6 0.6 msec, in

CGP 6.9 6 2.5 msec, P ¼ 0.34; decay tau in ACSF 19.0

6 7.0 msec, in CGP55845 16.2 6 10.1 msec, P ¼ 0.46)

or at �50 mV (n ¼ 3; 10–90% rise time in ACSF 11.6 6

4.2 msec; in CGP55845 11.66 4.0 msec; P¼ 0.98).

Unitary GABAA kinetics were measured by using one

presynaptic action potential with the postsynaptic GC

held in voltage clamp at �90 mV. Responses generated

Figure 2. Extent of dentate NGFCs within and beyond the molecular layer. A: Example of a subiculum-projecting NGFC (reconstructed in

C), in which the locations of the soma, dendritic tree, and axonal arbor were measured and plotted onto an arc of the molecular layer (ML).

The arc was flattened and divided into outer (O; top), middle (M), and inner (I; bottom) layers to produce an overview map of single NGFCs:

locations of cell bodies (red dots; n ¼ 8 in MML, n ¼ 9 in OML), dendritic fields (green circles), and axonal clouds (purple boxes). Sub, subic-

ulum; GCL, granule cell layer. The presence of axons within the subiculum is indicated by a plus sign to the right of the map. B: Plots of

16 additional NGFCs in the dentate, constructed as shown in A. The axonal arbors covered, on average, 37.7% 6 7.5% of the total length of

the molecular layer, which was, on average, 1,122.9 6 3.9 lm in length. Note that 11 of the 17 measured cells had axons projecting across

the hippocampal fissure into the subiculum or CA1 regions (indicated by a plus sign to the right of the map). C: Camera lucida reconstruction

of the subiculum-projecting NGFC mapped in A. Soma and dendrites are in green, axon in purple. Scale bars ¼ 100 lm.

Armstrong et al.


by NGFCs (Fig. 4A) were characteristically slower (n ¼ 7;

NGFCs 10–90% rise time 5.8 6 1.1 msec, decay time

constant 14.7 6 3.6 msec) and much smaller in ampli-

tude (NGFC amplitude �8.01 6 1.22 pA) than responses

generated by PV-positive fast spiking basket cell (PVBC)

to granule cell connections (Fig. 4B; n ¼ 5 PVBCs; 10–

90% rise time 0.99 6 0.12 msec, decay time constant

4.1 6 0.7 msec, amplitude �46.12 6 15.41 pA). How-

ever, although the amplitude of the unitary response to

PVBCs at the soma was more than 475% greater than the

NGFC response (Fig. 4C; P ¼ 0.01), the overall charge

transfer of the PVBC response was comparable to the

NGFC response (area under IPSC curve for PVBCs

�237.5 6 81.2 pAms, for NGFCs �140.1 6 29.4 pAms;

P ¼ 0.23), underscoring the importance of the prolonged

nature of the inhibition generated by NGFCs.

Inputs to dentate NGFCsFewer spontaneous events were observed in NGFCs

(5.3 6 0.6 Hz; n ¼ 6) held in voltage clamp at �80 mV in

control ACSF compared with dentate granule cells

(Fig. 5A,B; 12.9 6 2.2 Hz; n ¼ 6; P < 0.01), which is con-

sistent with their smaller dendritic fields. When the GABA

receptor antagonists gabazine and CGP55845 were

washed into the bath, the spontaneous event frequency

in NGFCs was reduced by 31.9% 6 7.8% (P ¼ 0.02),

revealing an sEPSC frequency in these cells of 3.6 6 0.5

Figure 3. NGFC output to GCs. A: Characteristic firing patterns of a

dentate NGFC (top traces) and GC (bottom traces) showing the NGFC’s

slight depolarizing ramp with late firing and unique afterhyperpolarization

shape. B: Average postsynaptic current responses in granule cells voltage

clamped at �50 mV (middle trace, n ¼ 9 pairs) or �90 mV (bottom

trace, n¼ 7 pairs) following a single action potential in presynaptic NGFCs

(top trace). Note that the early ionotropic GABAA component is outward at

�50 mV and inward at �90 mV and that the slow metabotropic GABABcomponent is not visible at�90 mV, close to its reversal potential. C: The

late GABAB component can be abolished by the GABAB antagonist

CGP55845, as demonstrated in averaged traces (n¼ 3 pairs), normalized

to control GABAA peak (traces organized as in B). D,E: The GABAB antago-

nist CGP55845 has no effect on the amplitude of GABAA currents (P ¼0.16), but the GABAB component of the postsynaptic response (measured

as area under the GABAB curve after the GABAA response has returned to

baseline) is abolished by the CGP55845 (*P ¼ 0.02). [Color figure can be

viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 4. NGFC vs. PVBC responses in GCs. Average currents in

GCs held in voltage clamp at �90 mV (bottom traces) elicited by

a single action potential (top traces) in NGFCs (n ¼ 7 pairs; A) or

PVBCs (n ¼ 5 pairs; B) demonstrate the significant difference in

the amplitude of the responses measured at the GC soma (*P ¼0.01; C). Note that, because of the prolonged nature of the

response to NGFCs, the inhibitory charge transfer, as measured

by the area under the response at �90 mV, is comparable (see

text; P ¼ 0.30).



Figure 5. Inputs to dentate NGFCs. A: Example traces of sPSCs in an NGFC (top trace) and a GC (bottom trace). B: Quantification of

sPSCs in GCs (n ¼ 6) and NGFCs (n ¼ 6), demonstrating that NGFCs receive significantly fewer spontaneous inputs than GCs (*P <

0.01). sPSCs were further separated into excitatory (EPSC; n ¼ 6; 3.6 6 0.5 Hz) and inhibitory (IPSC; n ¼ 4; 2.6 6 0.5 Hz) events using

the GABA antagonists CGP55845 and gabazine or the glutamate receptor antagonists NBQX and D-APV, respectively. C: Experimental

setup for field stimulation experiments. A stimulating electrode was placed on the subicular side (Sub) of the hippocampal fissure to stim-

ulate the perforant path, and a patch pipette was used to record responses in molecular layer (ML) NGFCs. GCL, granule cell layer; P, pos-

terior; A, anterior; M, medial; L, lateral. D,E: An inward current mediated primarily by AMPA channels was observed in NGFCs in response

to perforant path stimulation. Specific antagonists were bath applied sequentially, beginning with GABA antagonists CGP55845 and gaba-

zine, followed by the NMDA antagonist D-APV, and finally by the AMPA antagonist NBQX. Quantification (D) and an example (E) of wash-in

data are presented. D: Quantified data were normalized to response amplitudes in normal ACSF. Perforant path input was not blocked by

GABA antagonists (CGP55845 and gabazine; n ¼ 8; P ¼ 0.49) but was abolished by glutamate antagonists (NBQX and D-APV; n ¼ 10;

*P < 0.01). E: In the example traces, the stimulation artifact (truncated) is followed by an inward current that persisted in GABA and

NMDA antagonists but was abolished by the AMPA antagonist NBQX. F,G: Perforant path input exhibits facilitation at 50 msec, but not

longer interstimulus intervals (ISIs). Paired-pulse ratio was expressed as the second peak:first peak amplitude. F: Example traces are aver-

aged responses in a single NGFC to paired pulses of varying ISIs (overlaid). G: Quantification of paired perforant path stimulation reveals a

significant facilitation at 50 msec ISI (n ¼ 5; *P < 0.01) but not at 100 (n ¼ 5, P ¼ 0.17), 150 (n ¼ 5, P ¼ 0.54), or 200 msec (n ¼ 5;

P ¼ 0.93) ISIs. Scale bar ¼ 200 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Armstrong et al.


Hz. To examine specifically the rates of sIPSCs in NGFCs

compared with GCs, a CsCl-based internal solution with a

high [Cl–] was used in conjunction with the glutamate re-

ceptor antagonists D-APV and NBQX. Under these condi-

tions, the rate of sIPSCs in NGFCs was 2.6 6 0.5 Hz (n ¼4), still significantly less than sIPSC frequency in GCs

(9.16 1.9 Hz; n ¼ 8; P ¼ 0.04).

To examine specific inputs to NGFCs, field stimulation

of perforant path fibers on the subicular side of the hippo-

campal fissure was performed (Fig. 5C). Perforant path

stimulation induced an inward current in dentate NGFCs

held in voltage clamp at �75 mV (Fig. 5D,E; n ¼ 11). This

current was not blocked by sequential wash-in of the

GABA antagonists gabazine and CGP55845 (P ¼ 0.49)

nor the NMDA antagonist D-APV (P ¼ 0.09) but was abol-

ished by the AMPA antagonist NBQX (Fig. 5D,E; P < 0.01;

amplitudes, normalized to amplitude in normal ACSF, in

gabazine and CGP55845 97.0% 6 9.3%, n ¼ 8; in GABA

antagonists þ D-APV 71.1% 6 13.2%, n ¼ 4; in GABA

antagonists þ D-APV þ NBQX 9.0% 6 2.9%, n ¼ 10). In a

subset of the experiments (n ¼ 3), partial washout of

NBQX was performed to ensure that perforant path

evoked responses would reappear (washout required

�15 minutes). Paired-pulse stimulation of the perforant

path resulted in facilitation with a 50-msec ISI (Fig. 5F,G;

n ¼ 5; PP ratio ¼ 1.26 6 0.05; P < 0.01) but not at lon-

ger ISIs (Fig. 5F,G; PP ratio at 100 msec ¼ 1.15 6 0.09,

P ¼ 0.17; 150 msec ¼ 0.97 6 0.05, P ¼ 0.54; 200 msec

¼ 1.016 0.13, P ¼ 0.93).

NGFCs form diverse synapses withdiverse cells

Finally, we investigated the local microcircuits in which

NGFCs participate within the molecular layer. For all

interneuronal connections, at least one cell of the pair

was recovered and identified as an NGFC by using axonal

morphology. NGFCs form both chemical and electrical

connections with other interneurons, so we tested pairs

for each type of connection. Chemical synaptic con-

nections had longer durations and delays to peak of

>3 msec, whereas electrical connections had shorter

durations, had delays to peak of <3 msec, and could also

be observed by injecting a hyperpolarizing current into

the presynaptic cell, resulting in an outward current in

the postsynaptic cell. Among the recorded confirmed

NGFC to interneuronal pairs, 11 of 14 (79%) were con-

nected. All possible two-cell connection motifs were

observed (Fig. 6A), including synaptic only (Fig. 6B,C; n ¼3), electrical only (Fig. 6D; n ¼ 4), and both synaptic and

electrical (Fig. 6E,F; n ¼ 4). NGFCs were repeatedly

observed to form electrical synapses with other types of

molecular layer interneurons (n ¼ 6), which were con-

firmed as non-NGFCs by firing pattern and/or morphol-

ogy (Fig. 6C,D). The electrical coupling coefficient (the

percentage of the current injected at the presynaptic

soma that could be observed as an outward current at

the postsynaptic soma) in these pairs was 3.85% 6

0.89% on average [comparable to coupling coefficients

observed in animals of similar ages in various brain areas

and cell types (Amitai et al., 2002; Meyer et al., 2002;

Price et al., 2005; Simon et al., 2005)]. Among the 11

total pairs, both neurons could be positively identified as

either NGFCs or non-NGFCs in nine pairs; two pairs were

between two NGFCs, and seven pairs were between

NGFCs and non-NGFCs. Some examples and a summary

of the diversity of connectivity of dentate NGFCs

observed in these experiments are given in Figure 6.

DISCUSSION

Cells of the NGFC family (including both NGFCs and ivy

cells) have emerged as major players in the microcircuitry

of many brain areas, providing a unique type of slow but

powerful inhibition and forming connections with numer-

ous and diverse neuronal types. Estimates suggest that

nearly 40% of interneurons in the hippocampus are NPY-

and nNOS-positive cells that are likely a part of this family

(Fuentealba et al., 2008). Neurogliaform family cells have

been hypothesized to play important roles in modulating

spike timing, neuronal synchrony, generation of oscilla-

tions, strength of feedforward inhibition, tonic inhibition,

and perhaps even neurovascular control through release

of NO (Tamas et al., 2003; Simon et al., 2005; Zsiros

et al., 2007; Fuentealba et al., 2008; Price et al., 2008;

Olah et al., 2009; Karayannis et al., 2010).

Here we have described NGFCs in the dentate gyrus

for the first time. We have characterized their intrinsic

properties, including their hyperpolarized membrane

potential and characteristic late spiking firing pattern.

The anatomical data revealed NGFC axons that covered a

wide area of the middle and outer molecular layers, even

extending into other brain areas such as subiculum and

CA1 (similar to Ceranik et al., 1997), suggesting that

these cells may have a role in coordinating activity of

other cells within large sections of the dentate gyrus, and

even between regions, providing direct GABAergic links

between the dentate gyrus and the CA1 and subicular

regions. Because of the heterogeneous nature of their

synaptic partners, and because of their putative ‘‘volume

transmission’’ in which any dendrite passing through the

uniquely dense NGFC axonal cloud should receive NGFC

input, we expect that NGFCs would be likely to synapse

onto a variety of cell types with dendrites passing within

their axonal clouds, both in the dentate molecular layer

and in the CA1 and subicular regions. In addition, we



confirmed the presence of several molecular markers of

NGFCs in our recorded cells: NPY, COUP-TFII, nNOS, and

reelin. We examined the output of molecular layer NGFCs

to GCs and observed that the responses were mediated

by both GABAA and GABAB currents, with the former

being slower and smaller in amplitude but with charge

transfer comparable to GABAA responses evoked by

PVBCs. We noted that NGFCs receive low-frequency

Figure 6. Specific examples of the diverse interneuronal connections of NGFCs. A: Several different connectivity motifs were observed

between NGFCs and other molecular layer (ML) interneurons, either other NGFCs or non-NGFCs. In each case, at least one cell of the pair

(in each case, shown in dark green) could be positively identified as an NGFC. Each observed motif is lettered B–F, and B–F represent

examples corresponding to these letters. The numbers of observations of each motif were as follows: B, n ¼ 2; C, n ¼ 1; D, n ¼ 4; E, n

¼ 1; F, n ¼ 3. B: Bidirectional synaptic connection from NGFC to non-NGFC (B1) and non-NGFC to NGFC (B2). C: Unidirectional NGFC to

molecular layer interneuron synaptic connection. D: Camera lucida reconstruction of an electrically connected NGFC to non-NGFC pair

(0.6% coupling coefficient, electrophysiology not shown), demonstrating heterologous electrical coupling between two distinct cell types.

NGFC soma and dendrites are in green, NGFC axon in purple, non-NGFC soma and dendrites in blue, and non-NGFC axon in red. Reference

lines represent the outer edge of the molecular layer and the borders of the granule cell layer (GCL). E: Camera lucida reconstruction (E1)

and electrophysiology (E2–6) from an electrically and synaptically connected NGFC to non-NGFC pair (color scheme and scale bar as in D,

non-NGFC axon not recovered). Electrophysiology: firing pattern of NGFC (E2; dark green, left) and non-NGFC (E3; light blue, right); NGFC

(green; E4) to non-NGFC (blue) chemical synaptic connection; non-NGFC to NGFC chemical synaptic connection (E5) and electrical connec-

tion (E6); electrical connections revealed by injecting a �100-pA hyperpolarizing current into one presynaptic (Pre) cell (top, blue) and observ-

ing an outward current in a postsynaptic (Post) cell (bottom) held in voltage clamp at �75 mV. Electrical coupling coefficient for this pair

was 2.3% (see text). F: Example of a NGFC to non-NGFC pair with an electrical coupling coefficient of 7.8% and a unidirectional chemical syn-

aptic connection. The electrical connection was revealed bidirectionally (F1,2) as described for E. For chemical synaptic connections (F3,4)

single or dual action potentials were elicited in the presynaptic cell (upper traces), eliciting (lower traces) an electrically mediated response

in the postsynaptic NGFC (F3; green), and both an electrically and chemical synaptically mediated current in the postsynaptic non-NGFC (F4;

blue). Note that, in this pair, the difference between electrical and chemical synaptic currents can be clearly seen as two nearly instant peaks

representing the electrical connection riding on the rise of the delayed, larger amplitude chemical synaptic current. Scale bars ¼ 100 lm.

Armstrong et al.


spontaneous events composed of both excitatory and in-

hibitory PSCs and that they receive input from the ento-

rhinal cortex through the perforant path. Finally, we

observed a number of types of electrical and chemical

connectivity motifs between NGFCs and other molecular

layer interneurons.

Identification of dentate NGFCsThe rigorous identification of interneuronal cell types

requires that many factors be taken into account, includ-

ing firing pattern, molecular markers, synaptic properties,

and morphology. NGFCs present a particular challenge

because few of these characteristics alone are definitive.

NGFCs characteristically exhibit a late-spiking firing pat-

tern, often with a ramp in membrane potential just before

firing, a relatively fast time constant, and no sag.

Although no sag is apparent in NGFCs of other brain

areas of rats, human (Olah et al., 2007), and monkey

(Povysheva et al., 2007), NGFCs do have a sag. Although

firing pattern was useful in identifying candidate NGFCs

for post hoc identification, it was insufficient to identify

NGFCs positively. Thus, firing pattern was used only to

select cells for further investigation.

Molecular markers are often helpful in confirming inter-

neuronal cell types (for example, basket cells can be

discretely segregated into PVþ and CCKþ subgroups,

making such markers extremely useful for identification).

As in other brain areas, we found that NGFCs in the den-

tate do express certain molecular markers, including

NPY, nNOS, COUP TFII, and reelin [other known markers

include a-actinin, GABAAa1, and GABAAd (Deller and

Leranth, 1990; Ratzliff and Soltesz, 2001; Price et al.,

2005; Simon et al., 2005; Karagiannis et al., 2009; Olah

et al., 2009; Fuentealba et al., 2010; Tricoire et al.,

2010)]. However, no single one of these markers appears

to be expressed by every NGFC, nor is any one of the

markers specific to NGFCs alone (Nusser et al., 1995;

Fuentealba et al., 2008; Karagiannis et al., 2009; Tricoire

et al., 2010), making it difficult to identify NGFCs on an

individual basis by using markers alone.

NGFC family cells have a unique ability to elicit a bipha-

sic postsynaptic response consisting of both GABAA and

GABAB currents with a single presynaptic action potential.

Based on this synaptic property, a robust GABAB response

to a single presynaptic action potential can be used to iden-

tify cells of the NGFC family positively. Indeed, we used

this property on some (n ¼ 2) occasions to identify NGFCs

(see below). However, methods requiring paired recordings

are generally impractical for identifying single NGFCs.

Filled NGFCs could be identified by recovering a suffi-

cient portion of the axonal arbor to confirm the character-

istic density and frequent boutons. Because this was the

most straightforward confirmation available, all single-cell

recordings of putative NGFCs were identified post hoc in

this manner. Such morphological assessment requires

high-quality filling, recovery, and DAB staining of these

cells, making cell identification one difficult aspect of

working with this cell type in the dentate. Investigators

wishing to study dentate NGFCs can identify them post

hoc primarily by their dense axons (multiple separate

branches of axon crossing a single plane of focus) with fre-

quent (2–3-lm interbouton interval) boutons and small

(tens of micrometers from the cell body) dendritic arbors.

Secondary characteristics that can be used to support the

identification of NGFCs include a late-spiking firing pattern;

molecular markers such as NPY, nNOS, COUP-TFII, reelin,

GABAAa2, GABAAd, and a-actinin; and slow GABAA and

GABAB responses evoked by single presynaptic spikes.

Paired recordings of dentate NGFCsThe synaptic properties of NGFCs presented a second

challenge to performing detailed studies. Because neuro-

transmission from NGFCs fatigues quickly in vitro with

repeated stimulation (Tamas et al., 2003), cells for paired

recordings were typically patched in a low-Ca2þ ACSF to

prevent the release of vesicles from NGFCs until electri-

cal control of the membrane potential could be achieved

(see also Szabadics et al., 2007). Under conditions of low

Ca2þ, the firing patterns of cells are altered and are thus

less reliable indicators of putative NGFCs for paired

recordings. Additionally, wash-in of normal ACSF (10

minutes) was required before a connection could be

tested, so paired recordings had to be extremely stable.

Once a connection had been established, NGFCs were

stimulated to fire an action potential only once every mi-

nute, again to prevent fatigue of the connection, limiting

the data acquired for each pair. If, during paired record-

ing, many action potentials were elicited in quick succes-

sion, the postsynaptic response gradually decreased in

amplitude and recovered only after tens of minutes.

Despite the challenges involved in rigorously identifying

and making paired recordings from dentate NGFCs, we

were able to record an ample number of both individual

cells and pairs for analysis.

Relationship of NGFCs to ‘‘MOPP cells’’Existing information on molecular layer interneurons

has included basket cells, axo-axonic cells, and even

migrating developing interneurons, all with different axo-

nal and dendritic morphologies (Ribak and Seress, 1983;

Han et al., 1993; Freund and Buzsaki, 1996; Morozov

et al., 2006; Amaral et al., 2007; Houser, 2007). However,

reference to the MOPP (molecular layer perforant path

associated) cell, a cell type first described by Han et al.

and Halasy and Somogyi, both in 1993, whose axons and

dendrites are located in the molecular layer, appears



often in the literature. Descriptions of molecular layer cell

morphologies have varied, including the cells that qualify

as MOPP cells (Ribak and Seress, 1983; Seress and

Ribak, 1983; Soriano and Frotscher, 1989, 1993; Halasy

and Somogyi, 1993; Frotscher et al., 1994; Deller et al.,

1996; Morozov et al., 2006; Amaral et al., 2007; Chitta-

jallu et al., 2007). Indeed, NGFCs are molecular layer

interneurons whose axons terminate in the same region

as the perforant path, qualifying them as MOPP cells, and

observations that some molecular layer interneurons

share properties with certain stratum lacunosum-molecu-

lare interneurons in CA1 (Khazipov et al., 1995; Nusser

et al., 1995; Vida et al., 1998; Price et al., 2005), later

determined to be NGFCs, suggest that at least some

previously described MOPP cells might have included

NGFCs. Additionally, previous reports of subiculum-

projecting outer molecular layer interneurons (Ceranik

et al., 1997) may in certain cases describe dentate

NGFCs. However, non-NGFCs fitting the description of

MOPP cells were also observed during this study, indicat-

ing that not all MOPP cells are NGFCs (Supp. Info. Fig. 1;

Halasy and Somogyi, 1993). We suggest that the term

MOPP cell be considered a broad category into which a

number of specific interneuronal subpopulations in the

molecular layer, including NGFCs, belong.

Additional reference has been made to GABAergic cells

with cell bodies in and around the GC layer, extensive

axonal arborization in the molecular layer. and innervation

of granule cell dendrites (MOLAX cells; Soriano and

Frotscher, 1993). In most cases, descriptions of MOLAX

cells are not consistent with the NGFCs described in this

study (Soriano and Frotscher, 1993; Frotscher et al.,

1994; Deller et al., 1996).

Inputs to and feed-forward inhibitoryrole of dentate NGFCs

To understand how these cells work in the context of

normal dentate circuitry, we wanted to gain some under-

standing of not only the targets of NGFC axons but also

the nature of their inputs. Perforant path (PP) stimulation

of NGFCs revealed that they do, indeed, receive input

from entorhinal cortex, and paired recordings of NGFC to

GC connections further revealed that these interneurons

can perform feed-forward inhibition. The fact that PP

inputs were facilitating at short (50 msec) but not longer

ISIs suggests that NGFCs may be more involved in situa-

tions in which the rate of input to the dentate is

increased, a property that could be important in regulat-

ing overall excitation or in coordinating timing among cell

groups. However, more investigation will be required to

determine how these cells respond to PP inputs in the

intact entorhinal–hippocampal circuit in vivo. NGFC stim-

ulation elicited both a slow GABAA as well as a late GABABcomponent in GCs, yielding a large inhibitory charge

transfer with even a single action potential. Indeed,

although the amplitude of PVBC-evoked events was more

than six times greater than events evoked by NGFCs, the

inhibitory charge transfer of the NGFC-evoked events was

still comparable to that of PVBC-evoked events, under-

scoring the distinct and potentially important inhibitory

role of the slow events evoked by NGFCs. The small size

of the response to NGFCs measured at the soma of GCs

may also be due in part to strong dendritic filtering owing

to the cable properties of GC dendrites (Soltesz et al.,

1995; Schmidt-Hieber et al., 2007), indicating that the

response is likely more robust in the GC dendrite. The

location of NGFC axons in the same dendritic compart-

ment in which PP input also terminates suggests that

these cells play a more important role in dendritic infor-

mation processing than at the soma. Thus, NGFCs may

help to establish or maintain the low excitability of GCs by

interacting directly with sources of incoming excitation.

Although the presence of GABAB currents in GCs in

response to repeated PP stimulation has been observed

(e.g., by Piguet, 1993), and GABAB receptors are prominent

in dentate GCs (Kulik et al., 2003), future investigations

into the dynamics of the PP!NGFC!GC pathway will

likely require PP stimulation of connected NGFC–GC

paired recordings as well as dendritic recordings from GCs

in which the filtering of NGFC inputs would be minimized.

Spontaneous events in NGFCs were infrequent com-

pared with those in GCs, a finding that is consistent with

their smaller dendritic arbors. Among these spontane-

ous inputs, NGFCs receive similar sEPSC and sIPSC fre-

quencies. Compared with NGFCs, GCs receive much

more frequent sIPSCs. Our findings are consistent with

the numerous local inhibitory inputs to GCs (Acsady et al.,

1998; Buckmaster and Dudek, 1999). However, both

NGFCs and GCs receive PP inputs in the intact brain. There-

fore, further studies will be necessary to understand exactly

how incoming PP excitation of NGFCs and GCs may interact

with events originating locally.

Molecular layer microcircuitsPaired recordings between NGFCs and other molecular

layer interneurons allowed us to make more specific

observations about the local connectivity of NGFCs in

this region. The two-cell motifs that we observed involv-

ing dentate NGFCs are particularly fascinating when

considering what the role of these cells might be in the

function of the dentate. Not only do NGFCs inhibit other

inhibitory neurons with chemical synapses, but they

have electrical connections to other types of inhibitory

interneurons, sometimes even concurrently. The effect

of having both electrical and chemical synaptic

Armstrong et al.


connections with other interneurons is not clear. Gap

junctions may serve to excite or inhibit nearby neurons

or may act as low-pass filters between cells and on

incoming inputs (Mitchell and Silver, 2003; Zsiros et al.,

2007). The diverse connectivity of these cells as demon-

strated by the existence of all potential two-cell motifs

might have a complex, major role in coordinating activity

among molecular layer interneurons.

In the dentate, the NGFC represents a unique type of

feed-forward inhibitory interneuron, receiving PP input

and subsequently inhibiting granule cells with biphasic,

slow inhibitory currents. Additionally, NGFCs have both

gap junction connections and inhibitory chemical synap-

ses with other molecular layer interneurons, both NGFCs

and non-NGFCs. Because of the seemingly opposing roles

of these two types of connections, NGFCs would be well

suited for inclusion in future computational studies of the

dentate (Santhakumar et al., 2005; Dyhrfjeld-Johnsen

et al., 2007; Morgan and Soltesz, 2008; Ferrante et al.,

2009) to help elucidate their functional significance. In

addition to their unique connectivity, the extensive nature

of NGFC axons within the molecular layer coupled with

the postulated ‘‘volume transmission’’ from these axons

(Olah et al., 2009; Capogna & Pearce, 2011) indicates

that dentate NGFCs could broadly modulate the overall

activity of the dentate and tune outputs of other inter-

neuronal classes. These cells could also play important

roles in pathological states. In addition, although the

number of hilar interneurons is preferentially diminished

in epilepsy and after head injury, molecular layer inter-

neurons are not lost (Buckmaster and Jongen-Relo, 1999;

Ratzliff and Soltesz, 2001), although some changes in

properties have been noted (Peng et al., 2004). In the

context of effects on GC excitability, both the loss of

feedback inhibition from hilar interneurons and the con-

tinued inhibition of other interneurons by NGFCs could

actually contribute to dentate gate breakdown in hyper-

excitable states. Computational methods will prove

invaluable for understanding what role NGFCs could play

in dentate gyrus microcircuits under pathological condi-

tions (Dyhrfjeld-Johnsen et al., 2007; Morgan and Soltesz,

2008). Dentate NGFCs are poised at the boundary

between incoming cortical input and GC output, where

their intrinsic and connective properties make them

unique components of the microcircuitry modulating

entorhinal–hippocampal interactions.

ACKNOWLEDGMENTS

We thank Rose Zhu and Dora Hegedus for expert

technical assistance and general encouragement, Dr.

Csaba Varga for advice on antibodies, and Dr. Stephen

Ross for the generous loan of a Nikon FN-1 Eclipse

microscope.

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