Structural and Functional
Organization of the Hippocampus
Hua-Tai Xu,1Zhi Han,2Peng Gao,1,3Shuijin He,1Zhizhong Li,1Wei Shi,1,3Oren Kodish,1Wei Shao,1,4Keith N. Brown,1,3
Kun Huang,5and Song-Hai Shi1,3,4,*
1Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA
2College of Software, Nankai University, 94 Weijin Road, Tianjin 300071, China
3Graduate Program in Neuroscience, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA
4Graduate Program in Biochemistry and Structural Biology, Cell and Developmental Biology, and Molecular Biology,
Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA
5Department of Biomedical Informatics, The Ohio State University, 333 West 10thAvenue, Columbus, OH 43210, USA
The hippocampus, as part of the cerebral cortex, is
essential for memory formation and spatial naviga-
tion. Although it has been extensively studied, espe-
cially as a model system for neurophysiology, the
cellular processes involved in constructingand orga-
we show that clonally related excitatory neurons in
thedeveloping hippocampus areprogressivelyorga-
nized into discrete horizontal, but not vertical, clus-
ters in the stratum pyramidale, as revealed by both
ysis with double markers (MADM). Moreover,distinct
from those in the neocortex, sister excitatory neu-
rons in the cornu ammonis 1 region of the hippocam-
pus rarely develop electrical or chemical synapses
with each other. Instead, they preferentially receive
common synaptic input from nearby fast-spiking
(FS), but not non-FS, interneurons and exhibit syn-
chronous synaptic activity. These results suggest
that shared inhibitory input may specify horizontally
clustered sister excitatory neurons as functional
units in the hippocampus.
The hippocampus together with the neocortex comprises most
of the cerebral cortex. Arising from the dorsal telencephalon
or the pallium, the hippocampus and the neocortex become
anatomically distinct parts of the cortex. The neocortex consists
of six layers of neurons, with excitatory neurons occupying
layers II to VI. In contrast, the hippocampus contains mostly a
single layer with densely packed pyramidal neurons (the stratum
pyramidale) that is divided into two major regions (cornu ammo-
nis 1 [CA1] and CA3) and a small transitional region (CA2). The
CA regions are capped by the dentate gyrus (DG) (Nauta and
Feirtag, 1986). As the most inferior part of the hippocampal for-
mation, the subiculum connects CA1 with the entorhinal and
Besides their structural differences, the circuit organization of
the hippocampus and the neocortex are also distinct. The thal-
amus relays incoming sensory input into the neocortex and
mainly targets layer IV neurons, which project up to the superfi-
cial layer II/III neurons. Layer II/III neurons project down to the
deep layer V and VI neurons, which project primarily out of the
neocortex, e.g., to the thalamus, brainstem, and spinal cord
(Douglas and Martin, 2004). On the other hand, the entorhinal
cortex (EC), located in the parahippocampal gyrus, provides
the major input to the hippocampus, either to the DG and the
CA3 regions or to the CA1 and the subiculum. The flow of infor-
mation within the hippocampus is mostly unidirectional, starting
in the DG, then moving to the CA3, the CA1, the subiculum, and
finally out of the hippocampus to the EC (van Strien et al., 2009).
Given that the hippocampus and the neocortex are derived
from neural progenitors expressing similar transcription factors
including Pax6 and Emx1/2 (He ´bert and Fishell, 2008), how
they adopt fundamentally different structural and functional or-
ganization, especially at the cellular level, remains an intriguing
Previous histological, genetic, and lineage-tracing studies
have provided a comprehensive understanding of the construc-
tion of the neocortex. Proliferation of neuroepithelial cells in the
neuroectoderm produces radial glial cells (RGCs), a transient
but pivotal cell population in neocortical development (Alvarez-
Buylla et al., 2001). With the cell bodies located in the ventricular
zone(VZ)lining theventricle, RGCsdisplaya bipolarmorphology
with one short apical process that reaches the luminal surface of
the VZ (i.e., the ventricular endfoot) and another long basal pro-
cess that extends to the pial surface (i.e., the radial glial fiber).
In addition to their well-characterized role in supporting radial
migration of newborn neurons (Hatten, 1990; Rakic, 1971),
RGCs are mitotically active and responsible for producing nearly
all neocortical excitatory neurons either directly or indirectly
1552 Cell 157, 1552–1564, June 19, 2014 ª2014 Elsevier Inc.
through transient amplifying progenitors, such as intermediate
progenitors (IPs, also called basal progenitors) (Anthony et al.,
2004; Englund et al., 2005; Haubensak et al., 2004; Malatesta
etal.,2000;Miyataetal.,2004; Noctoretal.,2001, 2004;Stancik
et al., 2010; Tamamaki et al., 2001). Newborn neurons then
migrate radially to constitute the future neocortex. Successive
waves of newly generated neurons migrate past the existing
early-born neurons and occupy more superficial positions,
creating neocortical layers in an ‘‘inside-out’’ fashion (Angevine
and Sidman, 1961).
Moreover, clonal analyses in the developing neocortex have
led to the ‘‘radial unit hypothesis’’ (Rakic, 1988). Interestingly,
we recently found that radially aligned sister excitatory neurons
preferentially form electrical synapses with each other, which
facilitates the development of specific chemical synapses
between sister neurons and the emergence of a functional
columnar organization in the neocortex (Li et al., 2012; Yu
et al., 2009, 2012). These studies demonstrate that clonal ana-
lyses of neuronal production and organization can provide
fundamental insights into the structural and functional develop-
ment of brain structures. To date, while the specifying signals
and patterning events of hippocampal development have been
extensively explored (Lee et al., 2000; Mangale et al., 2008; Niel-
sen et al., 2007; Tole et al., 1997; Xie et al., 2010; Zhao et al.,
and functional development of the hippocampus is still missing.
Previous lineage analyses of hippocampal development have
been limited to coarse embryonic studies using mouse chimeras
or mosaic transgene expression (Martin et al., 2002; Soriano
et al., 1995) and a retroviral study in rats showing that clonally
related neurons can befound in more than one cytoarchitectonic
region (Grove et al., 1992).
Inthisstudy,wegeneticallylabeled individualdividing progen-
itor cells in the VZ of the developing hippocampal primordium
using two different cell-type-specific approaches—retrovirus-
(MADM)—and investigated lineage-dependent structural and
functional organization of the hippocampus.
Hippocampal Pyramidal Neuron Clonal Clusters Labeled
by Retrovirus Infection
Similar to their counterparts in the neocortex, excitatory neurons
of the developing hippocampal primordium (Angevine, 1965;
Bayer, 1980; Nowakowski and Rakic, 1981), whereas inhibitory
interneurons originate distantly in the ventral telencephalon
(Pleasure et al., 2000; Tricoire et al., 2011). To selectively label
excitatory neuron progenitors, we took advantage of the exqui-
site fidelity of the subgroup A avian sarcoma and leukosis virus
(Brown et al., 2011; Seidler et al., 2008). Low-titer avian RCAS
(replication-competent ALSV long terminal repeat with a splice
acceptor) retrovirus expressing enhanced GFP (EGFP) was in-
jected into the lateral ventricle of Emx1-Cre;R26LSL-TVAiLacZ/+
mouse embryos, in which ALSV receptor TVA was specifically
expressed in progenitors of the dorsal telencephalon with the
Cre-loxP strategy (Gorski et al., 2002; Seidler et al., 2008). Injec-
tions were made on embryonic day 11 (E11), E12, and E14, and
brains were recovered at postnatal day 7 (P7) to P30 for serial
sectioning, immunohistochemistry, and 3D reconstruction to
recover all the labeled cells in the hippocampus.
Discrete clusters of labeled pyramidal neurons with charac-
teristic morphology were frequently observed (Figure 1A), sug-
gesting that spatially isolated neuronal clusters originate from
individual dividing progenitors and arethereby clones. To ensure
reliable identification of ‘‘true’’ individual clones, we serially
diluted the injected retrovirus to label on average just about
one excitatory neuronal cluster per hippocampus (Figures 1A–
1C; Movie S1 available online). Similar hippocampal excitatory
neuron clonal clusters were also readily labeled using Molo-
ney murine leukemia retrovirus (Figures 1D and 1E). To further
confirm theclonal relationshipbetween neurons withinindividual
clusters, we injected a mixture of low-titer retroviruses express-
ing either EGFP or the red fluorescent protein mCherry and
examined the probability of observing spatially isolated clusters
containing cells expressing the same fluorescent protein versus
those containing cells expressing different fluorescent proteins
in the hippocampus. We found that nearly all spatially isolated
clusters (256 out of 260) were composed of neurons expressing
confirmed quantitatively bythe nearest-neighbor distance(NND)
analysis (Figure 1F). This clear segregation of neurons express-
ing EGFP or mCherry into individual clusters strongly argues
that neurons in individual clusters are clonally related siblings.
We next analyzed the size of individual clones labeled at
different embryonic stages (Figures 1G and 1H). A small fraction
(<20%) of clones labeled at E11 were composed of a single
neuron, while the rest had two or more neurons. The single
neuron clones represent retroviral integration into a postmitotic
neuron arising from asymmetric neurogenic division. On the
other hand, the large clones likely represent retroviral integration
into a self-renewing progenitor that undergoes symmetric
amplification division(s) and/or multiple rounds of asymmetric
neurogenic division. The progressive increase in the percentage
of single neuron clones and the concomitant decrease in the
percentage of large clones suggest that as development pro-
ceeds, the frequency of asymmetric division increases and the
frequency of symmetric division decreases in the developing
Hippocampal Pyramidal Neuron Clonal Clusters Labeled
To corroborate our findings with retroviral labeling, we exploited
the MADM system (Bonaguidi et al., 2011; Hippenmeyer et al.,
2010; Zong et al., 2005). To achieve selective labeling of excit-
atory neuron progenitors and their progeny (i.e., clones), we
used the Emx1-CreERT2mouse line (Kessaris et al., 2006). A sin-
gle dose of tamoxifen (TM) was administered to timed pregnant
Emx1-CreERT2;MADM dams at E10, E11, E12, or E13 via intra-
peritoneal injection. Brains were recovered and analyzed at
In the absence of TM treatment, we found no labeled cells (n =
5 brains). With TM treatment, we observed on average one
discrete cluster of excitatory pyramidal neurons in each
Cell 157, 1552–1564, June 19, 2014 ª2014 Elsevier Inc. 1553
Figure 1. Clonal Pyramidal Neuron Clusters in the Hippocampus Labeled by Retrovirus
(A) Confocal images of consecutive sections harboring a single pyramidal neuron clonal cluster in the hippocampus of a P30 Emx1-Cre;R26LSL-TVAiLacZ/+mouse
infected with RCAS-EGFP virus at E11. Scale bar, 100 mm.
(B) Projection image of the clone. Broken lines indicate the location of the clone. Scale bar, 200 mm.
(C) 3Dreconstructionimage oftheclone.Linesindicate thecontoursofthehippocampalsubregions(red,CA1;green, CA3;blue,DG).Triangles represent thecell
bodies of labeled pyramidal neurons and are approximately five times the actual cell body size. Similar symbols and displays are used in subsequent figures.
(D) 3D reconstruction image of a P30 hippocampus infected with a mixture of retroviruses expressing EGFP (green) or mCherry (red) at E12. Broken lines indicate
a green fluorescent neuron cluster (1) and a red fluorescent neuron cluster (2).
(E) Confocal images of consecutive sections harboring clone 1 (top) and 2 (bottom) in (D). Projection images are shown to the right. Broken lines indicate the
locations of the clones. Scale bars, 100 mm and 200 mm.
(legend continued on next page)
1554 Cell 157, 1552–1564, June 19, 2014 ª2014 Elsevier Inc.
hippocampus. As predicted, individual clusters contained either
a combination of green and red fluorescent neurons (Figures 2A,
2B, and S1A; Movie S3) or yellow fluorescent neurons (Figures
2C, 2D, and S1B; Movie S4). We did not observe any clusters
with a mixture of green/red neurons with yellow neurons (0 out
of 26). These results suggest that individual clusters are derived
from a single progenitor and thereby represent clones. The size
pendent approaches clearly demonstrate that sister excitatory
neurons originating from individual progenitors form spatially
isolated clusters in the stratum pyramidale of the hippocampus.
Notably, glial cells were also observed in some clones (Figures
2A, 2C, and S1, arrowheads), suggesting that progenitors in
the VZof thehippocampal primordium arecapable of generating
glia in addition to neurons.
Progressive Development of Hippocampal Pyramidal
nization, we examined the progressive development of embry-
onic clones. We carried out in utero intraventricular injections
of serially diluted retrovirus expressing EGFP at E12 to label
dividing progenitors in the VZ of the hippocampal primordium
at clonal density. At 24 hr after injection (i.e., E12–E13), labeled
cells were predominantly single and either resembled RGCs
with cell bodies in the VZ or were multipolar short-process cells
that comprised two cells: one RGC-like (arrow) and another
multipolar short-process cell (filled arrowheads) contacting
the radial glial process (open arrowheads) (Figure 3A, left). To
confirm the identity of RGC-like cells, we performed immunohis-
tochemistry and found that RGC-like cells were positive for Pax6
(Figure 3B, arrows), Nestin (Figure S2A), and brain-lipid-binding
protein (BLBP) (Figure S2B), the specific markers expressed by
radial glial progenitors (RGPs). Moreover, we found that RGC-
like cells were mitotically active, as they possessed condensed
DNA (Figure S2C) and incorporated bromodeoxyuridine (BrdU)
(Figure S2D). Additionally, we observed cells expressing Tbr2,
an IP cell marker, in clonal clusters (Figure 3C, open arrows).
Indeed, our live imaging analysis of individual clones revealed
RGP divisions at the VZ surface (Figure 3D, top; Movie S5)
and IP divisions away from the VZ surface (Figure 3D, bottom;
At 48 hr after injection (i.e., E12–E14), we observed radially
arrayed clones comprising an RGP (arrow) with one to three
daughter cells (2.7 ± 0.2 cells per cluster, n = 23; filled arrow-
heads) arrayed along the radial glial process (open arrowheads)
(Figure 3A). At 96 hr after injection (i.e., E12–E16), typical clones
consisted of an RGP and on average four to five daughter cells
(4.7 ± 0.3 cells per cluster, n = 37) that remained largely orga-
nized vertically. Some daughter cells at the top of the clusters
acquired the typical morphology of a developing hippocampal
pyramidal neuron, likely representing the earliest-born neu-
rons in the clone. Six days after injection (i.e., E12–E18), nearly
all daughter cells acquired the typical pyramidal neuron
morphology. Moreover, the daughter cells (filled arrowheads)
no longer arrayed vertically along the radial glial process (open
arrowheads); instead, they became horizontally organized (i.e.,
parallel to the VZ surface, broken lines) in the future stratum pyr-
amidale. Interestingly, coinciding with this spatial organization
change, the radial glial processes of RGPs displayed a bending
Figure 2. Clonal Pyramidal Neuron Clusters in the Hippocampus
Labeled by MADM
(A) Confocal projection images of a single G2-X EGFP (green)/tdTomato (red)
clone labeled at E11. A high-magnification image (broken lines) is shown at the
bottom. Arrowheads indicate glial cells. Scale bars, 50 mm and 400 mm.
(B) 3D reconstruction image of the clone.
(C) Confocal projection images of a single yellow clone labeled at E10. A high-
magnification image (broken lines) is shown at the bottom. Scale bars, 50 mm
and 400 mm.
(D) 3D reconstruction image of the clone.
See also Figure S1.
(F) NND analysis of retroviral-labeled neurons. Colored lines represent the cumulative frequency of NNDs of the same color-labeled (EGFP/EGFP, green or
mCherry/mCherry, red) neurons, the different color-labeled (EGFP/mCherry, orange) neurons and the random data sets simulated 100 times (gray).
(G) Histogram of clonal cluster size labeled at different embryonic stages (E11, n = 35; E12, n = 198; E14, n = 27).
(H) Average size of clones labeled at different embryonic stages (**p < 0.01). Data are presented as mean ± SEM.
Cell 157, 1552–1564, June 19, 2014 ª2014 Elsevier Inc. 1555
near the future stratum pyramidale (Figure 3A, red open arrow-
heads). As time proceeded (E12–P1), the bend in the radial glial
process and the horizontal orientation of daughter cell distribu-
tion became more prominent. In comparison, neocortical clones
examined at the same developmental stages remained largely
vertical and there was little persistent bending in the radial glial
pocampal clones exhibit distinct developmental behavior from
neocortical clones in transforming from an initially vertical orga-
nization to a horizontal organization relative to the VZ surface.
Consistent with this, live imaging experiments showed that
below the bend of the radial glial process, the progeny of hippo-
campal RGPs were in close contact with the mother progenitor
and migrated radially along the radial glial process. However,
around the bend, the progeny progressively departed the
mother progenitor, acquired the pyramidal cell morphology,
dale (Figure S3B; Movie S7). Related to this, while neurons in
the neocortex exhibited a distinct birthdate-dependent inside-
out radial organization, neurons in the hippocampus did not
(Figure S2E). In addition, consistent with the fact that RGPs un-
dergo consecutive roundsof asymmetric division, sister neurons
within individual clones were not all born at the same time
To further confirm that sister pyramidal neurons are oriented
horizontally, but not vertically, in the stratum pyramidale, we
systematically characterized the spatial organization of clonal
clusters in the relatively mature hippocampus at P10–P30 (Fig-
ures 3E, 3F, and S3C). The vast majority of clones (72 out of
75) were indeed organized parallel to the stratum pyramidale.
Notably, the average size of clones labeled at E12 was similar
when examined at E16, E18, P1, or P10–P30 (E16: 4.7 ± 0.3,
n = 37; E18: 4.8 ± 0.5, n = 17; P1: 4.9 ± 0.5, n = 7; P10–P30:
4.8 ± 0.3, n = 143), suggesting that hippocampal pyramidal
neuron neurogenesis is largely complete by E16. Moreover,
campus are complete and the overall dispersion of clonally
related pyramidal neurons is limited.
No Preferential Electrical or Chemical Synapse
Formation between Sister Pyramidal Neurons in CA1
The clustering of sister pyramidal neurons in the hippocampus
raises the intriguing possibility that specific synapses are
formed between them, as observed in the neocortex (Yu et al.,
2009, 2012). To address this, we performed quadruple whole-
cell patch-clamp recordings to simultaneously record from
two EGFP-expressing sister pyramidal neurons and two nearby
non-EGFP-expressing pyramidal neurons in the stratum pyrami-
dale of the CA1 region (Figures 4A–4D). Clones were labeled at
E12 and recordings were performed at E17–P33.
Figure 3. Development of Hippocampal Pyramidal Neuron Clones
(A) Images of spatially isolated hippocampal clones 1 day (E12–E13),
2 days (E12–E14), 4 days (E12–E16), 6 days (E12–E18), and 8 days (E12–P1)
after EGFP-expressing (green) retrovirus injection at E12 stained with
DAPI (blue). Arrows indicate the RGC, and filled arrowheads indicate short-
process daughter cells of individual clones. Open arrowheads indicate
the long radial process. Broken lines indicate the VZ surface. Scale bars,
(B) Images of an E16 EGFP-expressing hippocampal clone (green) labeled at
E12 and stained with the antibody against Pax6 (red), an RGP marker, and
DAPI (blue). Arrows indicate the RGC that is positive for Pax6 and possesses a
short ventricular endfoot and a long radial process (open arrowheads). Filled
arrowheads indicate the short-process daughter cells arrayed along the radial
glial fiber. High-magnification images (broken lines) are shown to the right.
Scale bars, 50 mm and 10 mm.
(C) Images of an E16 EGFP-expressing hippocampal clone (green) labeled at
E12 and stained with the antibody against Tbr2 (red), an IP marker, and DAPI
(blue). Open arrows indicate the IP that is positive for Tbr2 and arrows indicate
the RGP. High-magnification images (broken lines) are shown to the right.
Scale bars, 50 mm and 10 mm.
(D) Time-lapse imaging analysis of RGP (top, arrows) and IP (bottom, open
arrows)divisions atand away from the VZ surface,respectively. Time (h:min) is
shown at the top. Arrowheads indicate the progeny. Broken lines indicate the
VZ surface. Scale bars, 20 mm.
(E) Image of a P20 clone labeled by retrovirus expressing EGFP (green) at E12
stained with DAPI (blue). The solid line indicates the midplane of the 3D clone
and the broken line indicates the plane of the stratum pyramidale contour. The
angle between the two planes (q?) reflects the spatial distribution of the clone.
Arrowhead indicates glial cells, and a higher-magnification image is shown in
inset. Scale bars, 50 mm and 25 mm.
(F) Quantification of the spatial distribution of clones with more than three
neurons in the stratum pyramidale of the mature hippocampus.
See also Figures S2 and S3.
1556 Cell 157, 1552–1564, June 19, 2014 ª2014 Elsevier Inc.
We recorded a total of 215 pairs of sister pyramidal neurons
and found that only one was connected by electrical synapses
and three pairs were connected by chemical synapses (Figures
4E and S4A). Similarly, only 1 out of 400 nonsister (i.e., one
EGFP-expressing and one non-EGFP-expressing) pyramidal
neuron pairs was connected by chemical synapses, and none
were connected by electrical synapses. In addition, only 1 out
of 155 pairs and 3 out of 155 pairs of similarly situated non-
EGFP-expressing pyramidal neuron pairs were connected by
electrical and chemical synapses, respectively. Similar results
were obtained with sister pyramidal neurons labeled by MADM
(Figures S5A and S5B). Together, these results suggest that
sister pyramidal neurons in CA1 do not preferentially form elec-
trical or chemical synapses with each other. The overall local
synaptic connectivity rate between hippocampal pyramidal
neurons is very low, as shown in previous studies (Knowles
and Schwartzkroin, 1981; MacVicar and Dudek, 1981; Schmitz
et al., 2001).
Synchronous Synaptic Activity between Sister
Pyramidal Neurons in CA1
Interestingly, while sister pyramidal neuron pairs in the CA1 do
not preferentially develop synapses with each other, they ex-
hibited synchronous activity. Prominent spontaneous currents
were frequently detected in EGFP-expressing sister pyramidal
neurons and their nearby non-EGFP-expressing pyramidal neu-
rons (Figures S4B and S4C). However, unexpectedly, more syn-
chronous currents were observed between sister pyramidal
neuron pairs (Figure 5A, red asterisks). To quantitatively assess
this, we computed the cross-correlogram of spontaneous cur-
rents and found that there was a significant increase in the activ-
ity around 0 ms between sister (Figure 5B, red arrow and inset),
that sister pyramidal neurons preferentially exhibit synchronous
(within 1 ms) spontaneous activity.
Of 158 EGFP-expressing sister pyramidal neuron pairs from
P10–P33 mice, more than 40% (66 out of 158) showed robust
synchronous spontaneous activity (Figures 5C and S4D). In
contrast, only ?12% (37 out of 316) of nonsister pyramidal
neuron pairs and only ?12% (16 out of 135) of similarly situated
non-EGFP-expressing pyramidal neuron pairs showed similar
synchronicity. Intriguingly, this synchronicity in spontaneous
activity appeared to be developmentally regulated (Figure 5D).
While the rate of finding a synchronous pair of sister pyramidal
neurons was low prior to P10 (2 out of 20 at P0–P4 and one out
of seven from P5–P9), it increased dramatically around the
second postnatal week (11 out of 35 at P10–P13, and 16 out
of 35 at P14–P17) (Figure 5D, green bars), suggesting that
the second postnatal week is a critical period for the develop-
ment of synchronous activity between sister pyramidal neu-
rons. This time window coincides with the critical time window
of chemical synapse formation in the hippocampus (Fiala et al.,
1998). A small increase in nonsister pyramidal neuron syn-
chronous activity was also observed (Figure 5D, black bars).
Notably, synchronous activity was also preferentially found
between sister pyramidal neuron pairs labeled by MADM (Fig-
Common Inhibitory Synaptic Inputs between Sister
Excitatory Neurons in CA1
aptic activity, we performed pharmacological experiments using
tetrodotoxin (TTX, 1 mM), a Na+channel blocker that prevents
action-potential generation (Figure 6A). We found that TTX treat-
ment eliminated a vast majority of spontaneous activity; more-
over, sister excitatory neurons no longer exhibited synchronous
Figure 4. No Preferential Synaptic Connectivity between Sister
Pyramidal Neurons in CA1
(A) Images of a quadruple recording of two EGFP-expressing sister pyramidal
neurons (3 and 4, green) and two nearby non-EGFP-expressing pyramidal
neurons (1 and 2) in CA1 filled with Alexa 546-conjugated biotin (red). Scale
bar, 50 mm.
(C) Sample traces of action potentials and hyperpolarization (red traces) trig-
gered in the presynaptic neurons and responses (black traces) recorded in the
postsynaptic neurons. The bold traces represent the average and the gray
traces represent individual recordings. Scale bars, 50 mV, 20 pA, and 100 ms.
(D) High-magnification image of morphological reconstruction of neurons
recorded in (A).
(E) Quantification of the rate of synaptic connectivity.
See also Figure S4.
Cell 157, 1552–1564, June 19, 2014 ª2014 Elsevier Inc. 1557
activity (Figures 6B and 6C). These results suggest that synchro-
nous activity between sister pyramidal neurons are synaptic
events triggered by spontaneous action potentials.
Pyramidal neurons in CA1 receive both glutamatergic excit-
atory and g-aminobutyric acid (GABA)-ergic inhibitory synaptic
inputs. To uncover the nature of synchronous activity, we took
advantage of the distinct reversal potentials of glutamate re-
ceptor-mediated excitatory postsynaptic currents (EPSCs)
and GABA-A receptor-mediated inhibitory postsynaptic cur-
rents (IPSCs). We identified sister pyramidal neuron pairs that
exhibited robust synchronous activity when neurons were
clamped at ?70 mV (Figures 6D–6F). As predicted, when the
same neurons were clamped at ?30 mV, both outward and
inward currents were observed (Figure 6G). Interestingly, only
outward, but not inward, currents exhibited clear synchroni-
zation (Figures 6H and 6I), suggesting that synchronized
synaptic activity is mediated by GABA-A, but not glutamate,
To further confirm this, we examined the effects of bicucul-
line (10 mM), a specific GABA-A receptor inhibitor, and D-APV
(50 mM) and NBQX (5 mM), the inhibitors of N-methyl-D-
aspartate (NMDA)-type and a-amino-3-hydroxy-5-methyl-4-
isoxazolepropionic acid (AMPA)-type glutamate receptors,
respectively. As expected, bicuculline treatment completely
Figure 5. Preferential Synchronization of
Spontaneous Activity between Sister Pyra-
midal Cells in CA1
(A) Sample traces of spontaneous activity of two
EGFP-expressing sister pyramidal neurons (1 and
4, green) and two nearby non-EGFP-expressing
pyramidal neurons (2 and 3, black). High-tempo-
ral-resolution displays of a segment of recordings
(thick line) are shown at the bottom. Red aster-
isks and thin lines indicate synchronized events
between sister pyramidal neurons. Black asterisk
indicates synchronized events between non-
EGFP-expressing pyramidal neurons. Scale bars,
20 pA, 400 ms, and 100 ms.
(B) Normalized cross-correlogram (Z score) for
neuron pairs in (A). Bin size is 1 ms. Note that the
frequency of events is significantly increased
around 0 ms (red arrow and inset) for the sister
pyramidal neuron pair (1 versus 4, green), but not
for any other neuron pairs. The gray region in inset
corresponds to ?1 ms % Dt % 1 ms. Similar
symbols and displays are used in subsequent
(C) Percentage of pyramidal neuron pairs from
P10–P33 mice exhibiting synchronized sponta-
neous activity (***p < 0.0001; n.s., not significant).
(D) Percentage of pyramidal neuron pairs ex-
hibiting synchronized spontaneous activity at
different developmental stages (**p < 0.01; n.s.,
See also Figure S5.
eliminated the synchronous activity be-
tween sister pyramidal neurons (Figures
6J–6L). In contrast, D-APV and NBQX
had no significant effect (Figure S6).
Together, these results demonstrate that sister pyramidal neu-
rons exhibit synchronous GABA-A receptor-mediated inhibitory
FS Interneurons Selectively Provide Common Synaptic
Input to Sister Excitatory Neurons in CA1
A diverse group of inhibitory interneurons provides synaptic in-
hibition to excitatory neurons in the hippocampus (Freund and
Buzsa ´ki, 1996; Klausberger and Somogyi, 2008; McBain and
Fisahn, 2001). Synchronous synaptic inhibitory activity may
arise from common inhibitory synaptic input received by sister
pyramidal neurons. To test this, we explored the circuitry
mechanism underlying the synchronicity. Quadruple whole-cell
recordings were performed onto two EGFP-expressing sister
pyramidal neurons, one adjacent non-EGFP-expressing pyra-
midal neuron, and one nearby inhibitory interneuron at P14–
P30 (Figure 7A). The identity of recorded neurons as excitatory
pyramidal neurons or inhibitory interneurons was confirmed by
their firing properties (Figure 7B) and morphological reconstruc-
tions (Figures 7E and 7F). If an inhibitory interneuron coinner-
vates two or more pyramidal neurons, synchronous input
should be detected. Indeed, action potentials in a fast-spiking
(FS) inhibitory interneuron (cell 1) simultaneously elicited faithful
postsynaptic currents (blue arrows) in two EGFP-expressing
1558 Cell 157, 1552–1564, June 19, 2014 ª2014 Elsevier Inc.
sister pyramidal neurons (green cells 3 and 4), but not in the
adjacent non-EGFP-expressing control pyramidal neuron (cell
2) (Figures 7C and 7D).
Interestingly, we also observed a strong interneuron subtype
specificity in providing common synaptic input selectively to sis-
ter pyramidal neurons. Around 45% (25 out of 56) of FS interneu-
rons simultaneously innervated nearby sister pyramidal neuron
pairs, whereas only about 11% (7 out of 64) of non-FS interneu-
rons coinnervated nearby EGFP-expressing sister excitatory
neurons (Figures 7G, yellow bars, and S7). Moreover, these
shared inhibitory inputs from FS interneurons were specific to
sister pyramidal neuron pairs, as only 12.7% (9 out of 71) of FS
interneurons coinnervated nearby nonsister pyramidal neuron
pairs (Figures 7G, black bars, and S7). In addition, only about
16.7% (3 out of 18) of FS interneurons coinnervated nearby simi-
larly situated non-EGFP-expressing pyramidal neuron pairs. The
midal neuron pairs or non-EGFP-expressing pyramidal neuron
pairs was 7.1% and 12.5%, respectively. The overall connectiv-
ity between interneurons and pyramidal neurons was ?30.5%
(105 out of 344 pairs), which is slightly higher than previously re-
ported (Knowles and Schwartzkroin, 1981; Lacaille et al., 1987).
Together, these results suggest that sister excitatory neurons in
Figure 6. Synchronized Activity between
Sister Pyramidal Neurons Is Mediated by
Synaptic GABA-A Receptors
(A–C) Synchronized activity is blocked by TTX
treatment. (A) Sample traces of spontaneous
activity of a sister pyramidal neuron pair before
and after TTX treatment. Red asterisks indicate
correlogram before (red) and after (black) TTX
treatment. (C) Average Z score at Dt = 0 ms
before (red) and after (black) TTX treatment (n = 7;
**p < 0.01).
(D–I) Synchronized activity is reversed around
?36 mV. (D–F) Sample traces of spontaneous
activity (D), normalized cross-correlogram (E),
and average Z score at Dt = 0 ms (F) of sister
pyramidal neuron pairs recorded at ?70 mV
(n = 3). (G–I) Sample traces of spontaneous
activity (G), normalized cross-correlogram (H),
and average Z score at Dt = 0 ms (I) from the
same pairs of sister pyramidal neurons recorded
at ?30 mV. Note that outward (red), but not
inward (black), events are synchronized (n = 3;
*p < 0.05).
(J–L) Synchronized activity is blocked by bicu-
culline treatment. Sample traces of spontaneous
activity (J), normalized cross-correlogram (K), and
average Z score at Dt = 0 ms (L) of sister pyramidal
neuron pairs before (red) and after (black) bicu-
culline treatment (n = 5; **p < 0.01). Scale
bars, 20 pA and 100 ms. Data are presented as
mean ± SEM.
See also Figure S6.
the hippocampus preferentially receive
common inhibitory synaptic input selec-
tively from nearby FS, but not non-FS, in-
terneurons, which would contribute to the synchronous activity
of sister pyramidal neurons.
Here, we used two powerful and specific genetic labeling
methods, including avian retroviral labeling in conjunction with
mouse genetics and MADM, and performed a systematic and
quantitative clonal analysis of excitatory pyramidal neuron pro-
duction and organization in the developing hippocampus. We
found that progenitors in the VZ of the hippocampal primordium
are RGPs with characteristic morphological, molecular, and
mitotic features. Moreover, the composition, organization, and
development of hippocampal clones at early embryonic stages
are highly reminiscent of those of neocortical clones (Noctor
et al., 2001). However, as time proceeded, we began to observe
interesting differences in clonal organization between the hippo-
campus and the neocortex that have not been appreciated pre-
viously. While the daughter cells of neocortical clones remain
predominantly arrayed radially/vertically along the radial glial
process/fiber, those of hippocampal clones become progres-
sively horizontally distributed along the future stratum pyrami-
dale. This transition from a radial to horizontal organization of
Cell 157, 1552–1564, June 19, 2014 ª2014 Elsevier Inc. 1559
daughter cells is accompanied by a dramatic morphological
change in hippocampal, but not neocortical, RGPs. The radial
glial fiber of hippocampal RGPs exhibits a progressive bending
near the future stratum pyramidale. Interestingly, this bend in
the radial glial process has been noted previously in the fetal
monkey hippocampus (Nowakowski andRakic, 1979).Thebasis
of this bending remains to be determined and is likely linked to
the overall expansion and folding of the hippocampal formation.
Nonetheless, our data suggest that this special morphological
change likely facilitates the sequential deposition of the neuronal
progeny of individual RGPs along the stratum pyramidale and
the formation of a predominantly one-layered hippocampus. A
new mode of neuronal migration in the hippocampus has
recently been reported (Kitazawa et al., 2014) and may also
contribute to the horizontal organization of hippocampal pyrami-
As a consequence of this radial-to-horizontal shift in clonal or-
ganization, sister excitatory neurons in the mature hippocampus
Figure 7. FS Interneurons Selectively Provide Common Synaptic Input to Sister Pyramidal Neurons in CA1
(A) Images ofaquadruple recording oftwoEGFP-expressing sisterpyramidalneurons (3and 4,green), oneadjacentnon-EGFP-expressing pyramidal neuron(2),
and one nearby FS interneuron (1) filled with Alexa 546-conjugated biotin (red). Scale bar, 50 mm.
(B) Firing patterns of the four neurons in (A). Sample traces of neurons in response to current injection (bottom). Scale bars, 50 mV, 500 pA, and 200 ms.
(C) Sample traces of the connectivity between the four neurons. Blue arrows indicate postsynaptic responses in sister neurons 3 and 4 triggered by action
potentials in neuron 1 and orange arrows indicate reciprocal postsynaptic responses in neuron 1 triggered by action potentials in neuron 3. Scale bars, 50 mV,
20 pA, and 200 ms.
(D) Schematic of the connectivity among the four neurons.
(E and F) Morphological reconstruction images of the four neurons and the slice bearing them.
(G) Rate of dual connectivity between FS or non-FS interneurons and sister or nonsister pyramidal neurons (**p < 0.01; n.s., not significant).
See also Figure S7.
1560 Cell 157, 1552–1564, June 19, 2014 ª2014 Elsevier Inc.
are organized into discrete horizontal clusters, but not radial
arrays, as suggested previously (Martin et al., 2002). Our clonal
labeling of a single cluster in the entire hippocampus allows un-
equivocal characterization of the distribution of sister pyramidal
neurons. Clones exhibit considerable uniformity in distribution.
The spread of a clone is roughly proportional to the number of
neurons it contains. Similar observations have been made in a
previous retroviral study (Grove et al., 1992). However, another
study usingretroviruseswithdistinguishable tags has suggested
a wide spread of hippocampal clones (Reid et al., 1995). Our col-
lective observations of a single cluster withno scattered neurons
in individual hippocampi, no mixture of neurons originating from
differentially labeled progenitors, and similarly sized clonal clus-
ters observed at E16–E18 and P10–P30 do not support a wide
Importantly, hippocampal clones are distinct from neocortical
clones not only in structural organization but also in functional
organization. Previously, we found that vertically aligned sister
excitatory neurons in the neocortex preferentially develop elec-
trical and chemical synapses with each other (Yu et al., 2009,
2012), which contributes to the functional columnar organization
of the neocortex (Li et al., 2012). In contrast, despite their relative
region of the hippocampus is extremely sparse. Recent studies
suggest that the intraneocortical connections effectively amplify
thalamic input and thereby reinforce the sensory representation
(Li et al., 2013a, 2013b; Lien and Scanziani, 2013). On the other
hand, a limited local connectivity permits dynamic cooperation
of densely packed hippocampal pyramidal neurons for a large
number of representations and population coding (Klausberger
and Somogyi, 2008; Leutgeb et al., 2005).
In fact, GABAergic interneurons have been postulated to
contribute to the dynamic selection and control of cell assem-
blies in the hippocampus (Klausberger and Somogyi, 2008).
Remarkably, we found that even though sister pyramidal
neurons in CA1 are not directly synaptically connected, they
preferentially receive common synaptic input from nearby FS in-
terneurons.Related tothis,theyexhibitpreferential synchronous
spontaneous and evoked synaptic activity mediated by GABA-A
receptors. While additional mechanisms may contribute to syn-
chronous activity between sister pyramidal neurons, the exis-
tence of this precise local microcircuit is likely critical for sister
pyramidal neuron development and function. Moreover, FS in-
terneurons are powerful at controlling the timing and probability
of firing in pyramidal neurons (Hasenstaub et al., 2005). Previous
studies have demonstrated that in the hippocampus, common
inhibitory synaptic inputs can effectively entrain pyramidal
neuron activity and synchronize their firing via the interaction
of GABA-A receptor-mediated hyperpolarizing synaptic events
with intrinsic membrane conductance in pyramidal neurons
(Cobb et al., 1995; Hilscher et al., 2013). Unlike the neocortex,
in which correlated inhibitory synaptic activity between nearby
pyramidal neurons is common due to extensive connections
from interneurons to adjacent pyramidal neurons (Pfeffer et al.,
2013; Sippy and Yuste, 2013), synchronized inhibitory synaptic
events between nonsibling hippocampal pyramidal neurons
interneurons (Knowles and Schwartzkroin, 1981; Lacaille et al.,
1987; Lacaille and Schwartzkroin, 1988). We thus predict that
sister pyramidal neurons in the hippocampus are preferentially
synchronized in their activity, which will be fundamental for the
erential synchronization of sister excitatory neurons may allow
them to function as potential units to encode similar place fields
of hippocampal neurons with similar place fields have been
found in previous studies (Dombeck et al., 2010; Eichenbaum
et al., 1989; Hampson et al., 1999). It will be interesting to
test whether these clusters of functionally similar neurons are
indeed lineage related by exploiting recent advances in in vivo
functional imaging of hippocampal neurons at cellular resolution
in behaving animals (Dombeck et al., 2010; Ziv et al., 2013).
We also observed recurrent excitation from pyramidal neu-
rons to FS interneurons, suggesting a potential feedback loop
in synchronizing sister pyramidal neurons in the hippocampus.
The precise identity of FS interneurons that provide common
input selectively to sister pyramidal neurons remains to be
determined. Potential candidates include parvalbumin-positive
basket and axoaxonic cells located in the stratum oriens and
the stratum pyramidale (Kawaguchi et al., 1987; Sik et al.,
1995) that are capable of robustly driving synchronous activity
in pyramidal neurons (Cobb et al., 1995). Moreover, FS interneu-
rons exhibit specific firing patterns and contribute to different
aspects of network oscillations in vivo (Klausberger et al.,
2003; McBain and Fisahn, 2001). Therefore, they may control
sister pyramidal neurons in a temporally distinct and brain-
state-dependent manner (Gentet et al., 2010; Somogyi and
The mechanisms underlying the development of this lineage-
dependent fine-scale inhibitory connectivity remain to be inves-
tigated. FS interneurons in the hippocampus are produced in the
medial ganglionic eminence (Pleasure et al., 2000; Tricoire et al.,
2011). They arrive at the hippocampus at the embryonic stage
and occupy predominantly the stratum pyramidale and the stra-
tum oriens (Tricoire et al., 2011), where RGPs and newborn py-
ramidal neurons reside. It is possible that interactions between
local FS interneurons and nearby RGPs lead to the emergence
of preferential connectivity between FS interneurons and the
neuronal progeny of RGPs, which closely associate with the
mother RGPs during their production and initial migration. While
nectivity between sister pyramidal neurons, the formation of this
precise microcircuit is likely regulated by activity-dependent
In summary,we foundthat excitatory pyramidal neuronclones
in the hippocampus are organized into discrete horizontal clus-
ters. They preferentially share common inhibitory synaptic input
from nearby FS interneurons and exhibit synchronous activity. In
comparison, excitatory neuron clones in the neocortex are orga-
nized into discrete radial clusters. They preferentially develop
ties (Li et al., 2012; Yu et al., 2009, 2012). Together, these
findings demonstrate that the lineage relationship of excitatory
neurons fundamentally influences the structural and functional
organization of two different regions of the cerebral cortex with
Cell 157, 1552–1564, June 19, 2014 ª2014 Elsevier Inc. 1561
In Utero Infection and MADM Labeling
In utero intraventricular injection was performed as previously described (Yu
et al., 2009). For MADM labeling, Emx1-CreERT2;MADM-11GT/GTmice were
crossed with MADM-11TG/TGmice. Pregnant dams were injected intraperito-
neally with a single dose of tamoxifen (25–50mg/g body weight; Sigma) dis-
solved in corn oil (Sigma) at E10, E11, E12, and E13.
Serial Sectioning, Immunohistochemistry, Imaging, and 3D
Serial coronal or transverse sections (70 mm) of the brain were prepared using
a vibratome (Leica Microsystems) and processed for immunohistochemistry.
z stack images were taken using a confocal laser scanning microscope
(FV1000, Olympus) and were further analyzed using FluoView (Olympus) and
Photoshop (Adobe). For 3D reconstruction, each section was analyzed in
sequentialorderfrom rostraltocaudal usingNeurolucida andStereoInvestiga-
tor (MicroBrightField). Data are presented as mean ± SEM, and statistical dif-
ferences were determined using nonparametric Mann-Whitney-Wilcoxon and
Electrophysiology and Analysis
Transverse slices (250–350 mm) from E17–P33 mice were prepared using a vi-
bratome (Leica Microsystems) in choline solution bubbled with 95% O2/5%
CO2. Slices were kept in artificial cerebrospinal fluid (ACSF) at 32?C for 1 hr
and then at room temperature before recording. For recordings, slices were
transferred to a recording chamber perfused with warm bubbled ACSF at
32?C. Multiple electrodes of whole-cell recordings were made using an Axon
Multiclamp 700B amplifier and pCLAMP10.2 (Molecular Devices). Normalized
cross-correlograms of spontaneous events were analyzed as previously
described (Yu et al., 2012). Peaks in the cross-correlogram were considered
significant if individual bins exceeded any adjacent bins within 200 msby three
SDs (i.e., the difference of Z scores was >3). The statistical differences
between groups were determined by chi-square test.
figures,and seven movies and can be found with this article online at http://dx.
H.-T.X. and S.-H.S. conceived the project. Z.H. and K.H. performed quantita-
tive analysis on 3D datasets and helped with synchronization analysis. P.G.,
W. Shi, and O.K. prepared MADM samples. S.H. performed embryonic elec-
trophysiological recordings. Z.L. prepared retroviruses. W. Shao provided
S.-H.S. wrote the manuscript with comments from all of other authors.
We thank Drs. Nicoletta Tekki-Kessaris and Dieter Saur for providing the
Emx1-CreERT2and R26LSLTVA-iLacZmouse lines, Dr. Shaoyu Ge for sharing
retroviral vectors, and Dr. Eric Holland for initially supporting our avian retro-
viral engineering. We are grateful to Drs. Yasunori Hayashi, Alexandra L. Joy-
ner, Mary E. Hatten, and Mu-ming Poo and Ildiko Lily Erdy for critical reading
of the manuscript. This work was supported by grants from the NIH
(R01DA024681, R01MH101382, and P01NS048120) and the Simons Founda-
Received: October 21, 2013
Revised: February 21, 2014
Accepted: March 29, 2014
Published: June 19, 2014
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