10600 • The Journal of Neuroscience, July 22, 2015 • 35(29):10600 –10612

Development/Plasticity/Repair

Development of Adult-Generated Cell Connectivity with
Excitatory and Inhibitory Cell Populations in the
Hippocampus
Leonardo Restivo,1 Yosuke Niibori,1 Valentina Mercaldo,1 Sheena A. Josselyn,1,2,3,4 and Paul W. Frankland1,2,3,4
1

Program in Neurosciences and Mental Health, The Hospital for Sick Children, Peter Gilgan Centre for Research and Learning, Toronto, Ontario, Canada,
M5G 0A4, and Departments of 2Psychology and 3Physiology and 4Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada, M5S 1A8

New neurons are generated continuously in the subgranular zone of the hippocampus and integrate into existing hippocampal circuits
throughout adulthood. Although the addition of these new neurons may facilitate the formation of new memories, as they integrate, they
provide additional excitatory drive to CA3 pyramidal neurons. During development, to maintain homeostasis, new neurons form preferential contacts with local inhibitory circuits. Using retroviral and transgenic approaches to label adult-generated granule cells, we first
asked whether a comparable process occurs in the adult hippocampus in mice. Similar to development, we found that, during adulthood,new neurons form connections with inhibitory cells in the dentate gyrus, hilus, and CA3 regions as they integrate into hippocampal
circuits. In particular, en passant bouton and filopodia connections with CA3 interneurons peak when adult-generated dentate granulecells (DGCs) are ⬃4 weeks of age, a time point when these cells are most excitable. Consistent with this, optical stimulation of 4-week-old(but not 6- or 8-week-old) adult-generated DGCs strongly activated CA3 interneurons. Finally, we found that CA3 interneurons wereactivated robustly during learning and that their activity was strongly coupled with activity of 4-week-old (but not older) adult-generated
DGCs. These data indicate that, as adult-generated neurons integrate into hippocampal circuits, they transiently form strong anatomical,
effective, and functional connections with local inhibitory circuits in CA3.
Key words: adult neurogenesis; CA3; dentate gyrus; mossy fibers; optogenetics

Significance Statement
New neurons are generated continuously in the subgranular zone of the hippocampus and integrate into existing hippocampal
circuits throughout adulthood. Understanding how these cells integrate within well formed circuits will increase our knowledge
about the basic principles governing circuit assembly in the adult hippocampus. This study uses a combined connectivity analysis(anatomical, functional, and effective) of the output connections of adult-born hippocampal cells to show that, as these cells
integrate into hippocampal circuits, they transiently form strong connections with local inhibitory circuits. This transient increaseof connectivity may represent an homeostatic process necessary to accommodate changes in the excitation/inhibition balance
induced by the addition of these new excitatory cells to the preexisting excitatory hippocampal circuits.

Introduction
In the developing and adult hippocampus, new cells are generated in the subgranular zone (SGZ). The vast majority of thesecells differentiate into neurons and migrate into the granule cell
layer of the dentate gyrus (DG). They extend dendritic processes
Received Aug. 5, 2014; revised May 22, 2015; accepted June 20, 2015.
Author contributions: L.R., S.A.J., and P.W.F. designed research; L.R., Y.N., and V.M. performed research; L.R. and
P.W.F. wrote the paper.
This work was supported by Natural Sciences and Engineering Research Council of Canada Grant RGPIN312434
and Canadian Institutes of Health Research Grants MOP86762 (P.W.F.) and MOP74650 (S.A.J.). L.R. was supportedby the Hospital for Sick Children. We thank Chen Yan and Anne Wheeler for advice with the optogenetic experiments
and for statistical analysis, respectively. We also thank Shaoyu Ge for providing the ChIEF construct and A. de
Cristofaro, R. Braybon, and M. Yamamoto for technical assistance.

toward the molecular layer of the DG and mossy fiber axons
toward targets in the hilus and CA3 region, eventually forming
input and output synaptic connections (Zhao et al., 2006; Toni etal., 2007, 2008; Faulkner et al., 2008; Ide et al., 2008; Tronel et al.,2010; Deshpande et al., 2013; Sun et al., 2013). Although thissequence is invariable, integration of newly generated neurons
into hippocampal circuits proceeds at a slower rate during adulthood than during development (Duan et al., 2008).
Correspondence should be addressed to Paul W. Frankland, Program in Neurosciences and Mental Health, The
Hospital for Sick Children, Peter Gilgan Centre for Research and Learning, Toronto, Ontario, Canada, M5G 0A4.
E-mail: paul.frankland@sickkids.ca.
DOI:10.1523/JNEUROSCI.3238-14.2015
Copyright © 2015 the authors 0270-6474/15/3510600-13$15.00/0

Restivo et al. • Integration of Adult-Generated Hippocampal Neurons

Because the great majority of cells generated in the SGZ differentiate into excitatory neurons (Zhao et al., 2008), these cells
increase excitatory drive to target cells in CA3. There are several
possible ways in which neural circuits might accommodatechanges in balance between excitation and inhibition (Isaacson
and Scanziani, 2011; Jinde et al., 2012). In particular, in the hippocampus, new neurons may form connections with local inhibitory circuits in the DG, hilus, or CA3 (Meltzer et al., 2005; Ikrar
et al., 2013). For example, in the CA3 stratum-lucidum (CA3-sl),filopodial extensions from large mossy fiber terminals (LMTs)
contact inhibitory interneurons (Acsády et al., 1998), and thismay serve to maintain a balance between excitation and inhibition through feedforward inhibition (Meltzer et al., 2005; Danzer
et al., 2010). Indeed, in the postnatal hippocampus, as new dentate granule cells (DGCs) integrate, there is a transient increase infilopodial extensions from LMTs (Wilke et al., 2013). Similarly,
in the adult hippocampus, seizure induction promotes the formation of new filopodial extensions from LMTs (Danzer et al.,2010).
Accordingly, here we characterized the development of anatomical connections of newly generated DGCs in the adult hippocampus, focusing in particular on output connections withlocal inhibitory circuits. In particular, we asked whether changes
in connectivity mirror changes in excitability of newborn cells.
Because the excitability of adult-generated DGCs peaks between
4 and 6 weeks (Schmidt-Hieber et al., 2004; Ge et al., 2007; Mongiat et al., 2009; Gu et al., 2012; Marín-Burgin et al., 2012), we
predicted that the formation of anatomical connections with local inhibitory circuits should follow a similar time course. We
went on to ask whether optical stimulation of adult-generated
DGCs activates inhibitory interneurons in the hippocampus andwhether the activity of adult-generated DGCs and inhibitory interneurons is coupled during learning.

Materials and Methods
Mice
Wild-type mice. We used male mice derived from a cross between 129S6
and C57BL/6N mice (Taconic Farms).
nestin–CreERT2 ⫻ R26R–YFP mice. nestin–Cre ERT2⫹ mice express atamoxifen (TAM)-inducible form of Cre recombinase driven by a progenitor specific (nestin) promoter, as described previously (Imayoshi etal., 2008; Arruda-Carvalho et al., 2011). In these experiments, we used
line 4 from Imayoshi et al. (2008) because recombination efficiency ishighest in the SGZ of the hippocampus in this line (Imayoshi et al., 2008;
Arruda-Carvalho et al., 2011). These mice were crossed with R26R–yellow fluorescent protein (YFP) reporter mice (Lagace et al., 2007). In thisreporter line, YFP expression is under the control of the ubiquitous
Rosa26 locus promoter, and expression of the YFP transgene is dependent on the Cre recombinase-mediated removal of a transcriptional
STOP cassette. Accordingly, in male and female offspring from this cross,injection of TAM leads to expression of the YFP transgene only in nestinpositive (nestin ⫹) cells and their progeny. Both lines were maintained on
a C57BL/6N background. Genotypes were determined by PCR analysis of
tail DNA samples, as described previously (Lagace et al., 2007; ArrudaCarvalho et al., 2011).
Both wild-type (WT) and reporter lines were bred at The Hospital for
Sick Children. Mice were weaned at 21 d and housed three to five mice
per cage. They were maintained on a 12 h light/dark cycle (lights on at
7:00 A.M.) with access to food and water ad libitum. Behavioral procedures were conducted during the light phase of the cycle, blind to genotype of the mouse and according to protocols approved by the Animal
Care Committee at The Hospital for Sick Children.

Retroviral vectors
To characterize the morphology of new DGCs, we used a replicationdeficient retroviral vector (based on the Moloney murine leukemia virus)

J. Neurosci., July 22, 2015 • 35(29):10600 –10612 • 10601

in which a CAG promoter drives green fluorescent protein (GFP) expression (Tashiro et al., 2006; Stone et al., 2011). To stimulate new DGCs, weused a retroviral vector expressing a channelrhodopsin 2 chimera with acrossover site at loop E-F and Iisoleucine 170 mutated to valine (ChIEF)(Lin et al., 2009; Gu et al., 2012). Retroviruses were prepared in ourlaboratory by transfecting Plat-gp cells with plasmids containing an amphotropic envelope (vesicular stomatitis virus glycoprotein) and thetransgenes. For CAG–GFP virus, Plat-E cells were then infected to generate a stable virus-producing cell line. The concentrated virus solution
was obtained by ultra-speed centrifugation (average of ⬃1.7 ⫻ 10 9 infection units/ml).

Drugs
TAM (Sigma) was dissolved in minimal ethanol and suspended in sunflower seed oil (Lagace et al., 2007). Reporter mice received five daily
injections (180 mg/kg, i.p.), starting at postnatal day 42 (P42).

Surgical procedures
P10 and P42 WT mice were used for detailed morphological analyses ofmossy fibers. Adult mice (P42) were anesthetized with chloral hydrate(400 mg/kg, i.p) and placed in a stereotaxic surgical frame (ASI Instruments). The scalp was incised and retracted, and drill holes in the skull
were made above the hippocampi. Retrovirus expressing GFP was microinfused bilaterally into the DG [0.5 ␮l at a rate of 0.1 ␮l/min; coordinates: anteroposterior (AP), ⫺2.2 mm; mediolateral (ML), 1.8 mm;
dorsoventral (DV), 2.2 mm) via a glass micropipette. The micropipette
was left in place 5 min after the completion of the injection and thenremoved. The scalp wound was sealed with a stainless steel staple, andmice were treated with ketoprofen (5 kg/mg, s.c.) postsurgically.
Infant mice (P10) were anesthetized with a lower dose of chloral hydrate (200 mg/kg, i.p.) and placed in a modified stereotaxic frame toaccommodate their smaller skull. Retrovirus expressing GFP was microinfused bilaterally into the DG (0.2 ␮l via a glass micropipette). After the
microinfusion, the glass micropipette was removed immediately, and thewound was sutured. Mice were brought back to their home cage and left
undisturbed until the start of the experiment.
For the optogenetic experiments, adult mice were microinfused unilaterally with retrovirus expressing ChIEF, using procedures identical tothose described above. Five minutes after the completion of the microinfusion, the pipette was removed, the skull was gently scraped with ascalpel, and three anchoring screws were secured to the surface of theskull. One optical fiber [length, 2 mm; diameter, 200 ␮m; 0.39 numericalaperture (NA); Thorlabs] was lowered above the DG (coordinates: AP,⫺2.2 mm; ML, 1.8 mm; DV, 2.0 mm), ipsilateral to the injection site. The
optical fiber pedestal was fixed to the skull with dental cement.

Optical stimulation
Mice were transported to the experimental room in their home cage andreceived unilateral optical stimulation [two light ON epochs (20 Hz, 9
mW, and 15 ms pulse width for 180 s) separated by 3 min]. Previous
studies established that stimulation of DGCs at this frequency reliably
activates CA3 pyramidal cells (Mori et al., 2004) and induces Fos expression (Liu et al., 2012). The implanted optrodes were tethered to a laser
source (473 nm; Laserglow) through a split optic fiber (Precision Fiber
Products) and controlled by a function generator (Agilent Technologies). One minute after the offset of the light, mice were disconnectedfrom the laser source and returned to the vivarium. Ninety minutes after
the completion of the optical stimulation, mice were anesthetized andperfused.

Contextual fear conditioning apparatus and procedures

Contextual fear conditioning occurred in test chambers (31 ⫻ 24 ⫻ 21cm; Med Associates) with shock-grid floors (bars 3.2 mm in diameter
spaced 7.9 mm apart). The front, top, and back of the chamber were clear
acrylic, and the sides were modular aluminum. During training, mice
were placed in the chambers, and five footshocks (0.7 mA, 2 s duration, 1
min apart) were delivered after 2 min. Mice were removed from thechambers 1 min after the last shock. During training, mouse behavior
was monitored by video camera mounted on the ceiling of the cages, andthe time spent freezing (i.e., absence of all but respiratory movement;

Restivo et al. • Integration of Adult-Generated Hippocampal Neurons

10602 • J. Neurosci., July 22, 2015 • 35(29):10600 –10612

Kim and Fanselow, 1992) was recorded using an automated scoring system (Freeze Frame software; Actimetrics).

Fixation and tissue processing
Mice were anesthetized with chloral hydrate and perfused intracardiallywith ice-cold 0.1 M PBS, followed by 4% PFA. The brains were removed,
postfixed in 4% PFA, and stored at 4°C until processed further. Fifty
micrometer horizontal (for morphological analyses) or coronal (for behavioral and optogenetic experiments) sections were cut on a vibratome(VT1200s; Leica). Sections were washed in 0.1 M PBS and incubated inblocking solution (2.5% bovine serum albumin, 5% normal goat serum,
or 5% normal donkey serum, 0.3% Triton X-100) for 2 h. The following
primary antibodies were used: goat polyclonal anti-GFP (1:500; Invitrogen), mouse monoclonal anti-parvalbumin (1:1000; Sigma), chicken
polyclonal RFP (1:250; Millipore), mouse monoclonal anti-GAD67 (1:1000; Millipore), rabbit polyclonal anti-c-fos (1:1000; Calbiochem),mouse monoclonal anti-NeuN (1:1000; Millipore), and mouse monoclonal anti-Bassoon (1:200; Enzo Life Sciences). The slices were incubated for 2 h at room temperature with the following secondary
antibodies: Alexa Fluor 488 anti-rabbit, Alexa Fluor 488 anti-mouse,
Alexa Fluor 633 anti-mouse, Alexa Fluor 568 anti-chicken, and Alexa
Fluor 568 anti-rabbit (1:500; Invitrogen). The slices were washed in 0.1 M
PBS, counterstained with DAPI (1:1000; Sigma-Aldrich), mounted onslides, and coverslipped.

Specific experimental procedures and analyses
Morphological analyses. P42 mice were microinfused with retrovirus expressing GFP and then 2 (n ⫽ 4), 4 (n ⫽ 7), 6 (n ⫽ 6) or 8 (n ⫽ 7) weeks
later were perfused. Additionally, a group of mice (“developmental”; n ⫽7) received retroviral microinfusions at P10 and then were perfused 12
weeks later. Brains were sectioned horizontally to facilitate visualizationof mossy fiber projections to CA3.
Detailed morphological analyses were performed on high-resolution
stack images (objective, 63⫻/1.4 NA; voxel size, 0.07 ⫻ 0.07 ⫻ 0.8 ␮m;image size, 1024 ⫻ 1024 pixels) acquired with a confocal microscope(dorsoventral range, ⫺1.3 to ⫺2.5 mm; LSM 710; Zeiss). The hilus andthe CA3 region of the hippocampus were first identified using a lowmagnification objective (20⫻/0.8 NA). En passant boutons (defined as
regular-shaped axonal swellings lacking filopodia protrusions) wereidentified in both the hilus and CA3 region. LMTs (defined as irregularshaped axonal swellings and, in most cases, filopodia) were identified inCA3. LMT and en passant bouton area were measured in the z projectionof confocal stacks by contour tracing with FIJI (Schindelin et al., 2012).
Protrusions stemming from LMTs (1 ␮m ⬍ length ⬍ 20 ␮m; Acsády etal., 1998) with a bulbous head (cross-sectional area ⬍ 1 ␮m 2) were traced
and counted as filopodia (Danzer et al., 2010). Filopodia number was
expressed per LMT. Filopodia length and head size were measured in thez projection of confocal stacks by contour tracing with FIJI. Threedimensional reconstructions of LMTs were obtained from highresolution image stacks (objective, 63⫻/1.4 NA; voxel size, 0.02 ⫻ 0.02 ⫻0.46 ␮m; image size, 2048 ⫻ 2048 pixels) processed with an inverse
filtering deconvolution step (Image Pro; Media Cybernetics). Thesemorphological measures were conducted on GFP ⫹ axons that were atleast 200 ␮m in length. To calculate the density of LMTs and en passantboutons, longer (⬎500 GFP ⫹ ␮m) segments were analyzed. LMT andbouton density was expressed as number per 100 ␮m.
The number of infected cells (per DG) was quantified in a subset ofmice from each condition (n ⫽ 4 per group). The number of GFP ⫹ cells
was counted manually on Z-stack projections of horizontal sections (objective, 10⫻/0.3 NA; Z stack, five slices; voxel size, 1.38 ⫻ 1.38 ⫻ 6.24
␮m; image size: 1024 ⫻ 2048 pixels) and expressed as number of cells per1000 ␮m 2.
These and all subsequent analyses were performed by an experimenter
blind to the condition of the sample/animal.
Optogenetic experiments. WT mice were microinfused with retrovirus
expressing ChIEF and then 4, 6, or 8 weeks later received optical stimulation in their home cage. Ninety minutes later, they were perfused, andimmunohistochemistry for RFP, GAD67, and Fos was conducted on 50
␮m coronal sections (to facilitate identification of optrode tips). Under

low magnification (objective, 20⫻/0.8 NA; Z stack, 10 slices; voxel size,
1.38 ⫻ 1.38 ⫻ 3 ␮m; image size: DG, 1024 ⫻ 512 pixels and CA3, 512 ⫻512 pixels), the number of Fos ⫹ cells (expressed as number of cells per
square millimeter) and Fos ⫹/GAD67 ⫹ cells in DG and CA3 were quantified on both the illuminated and non-illuminated sides (dorsoventral
range, ⫺1.2 to ⫺2.5 mm). Seven animals were excluded from the analysis
because of either the absence of transgene expression or optical probe
misplacement (four from the 4 week group, three from the 6 week group,and one from the 8 week group). After these exclusions, sample sizes wereas follows: 4 weeks, n ⫽ 6; 6 weeks, n ⫽ 7; and 8 weeks, n ⫽ 7.
Induction of Fos in excitatory and inhibitory cell populations after contextual fear conditioning. P42 YFP reporter mice (n ⫽ 23) were treatedwith TAM and then 4 – 6 weeks later (from the first day of TAM injections) were trained in contextual fear conditioning. Ninety minutes after
contextual fear conditioning, mice were perfused, and Fos and GAD67
immunohistochemistry was conducted. To characterize activation in inhibitory cells, Fos expression was quantified in GAD67 ⫹ cells in the DG
and CA3. To characterize training-induced activation in putative excitatory cells, Fos expression was quantified in GAD67-negative (GAD67 ⫺)cells in the DG and CA3. Fos and GAD67 immunohistochemistry was
conducted similarly on tissue from age-matched home-cage control mice(n ⫽ 19). Fold increase in activation was computed by normalizing
counts in trained versus control mice for each region and cell population.
Slices were taken from the dorsal hippocampus (1 in 4 sampling from ⫺1
to 2.4 mm posterior to bregma), and sections from different groups were
processed simultaneously. Fos expression was quantified from ⫺1.2 to⫺2.5 mm in the dorsoventral range. Note that this corresponds to thesame dorsoventral range used for the morphological and optogenetic
analyses.
Correlated Fos expression in different cell populations after contextual
fear conditioning. In the same mice as above, we additionally conducted
immunohistochemistry for YFP. Then, to assess functional connections
among different hippocampal subpopulations, we evaluated to what degree Fos expression in adult-generated DGCs (i.e., Fos ⫹/YFP ⫹ cells)
covaried with Fos expression in other cell populations (e.g., Fos ⫹/GAD67 ⫹ cells in CA3) by computing pairwise correlations (Pearson’s r).
To minimize type I errors, significant pairwise correlations were further
probed using a permutation test (based on 9999 Monte Carlo resamplings). Functional connectivity may be inferred from within-subject
variance (which is typically the case in human imaging studies) or
between-subject variance (Horwitz et al., 1995; McIntosh, 1999). Because “activity” is inferred postmortem by changes in Fos expression(yielding single data point/mouse), here we computed covariance acrossmice to define functional connections between different neuronal subpopulations, as has been done previously (Soncrant et al., 1986; Fidalgo
et al., 2012, 2014; Wheeler et al., 2013).
Images were acquired with a confocal microscope under low magnification (objective, 20⫻/0.8 NA; Z stack, 16 slices; voxel size, 0.42 ⫻ 0.42 ⫻0.2 ␮m; image size: DG and hilus, 2048 ⫻ 1024 pixels and CA3, 1024 ⫻1024 pixels). Fos expression was quantified in adult-generated DGCs
(i.e., YFP ⫹ cells in the granule cell layer of the DG), as well as inhibitory(i.e., GAD67 ⫹) and putative excitatory (i.e., GAD67 ⫺) cells in the DG,
hilus, and CA3. Numbers of double-labeled cells (e.g., Fos ⫹/GAD67 ⫹ inCA3) were normalized with respect to total Fos expression in the hippocampus for each mouse. In a subset of slices, immunohistochemistry
for YFP and NeuN was conducted to assess maturation of YFP-tagged
cells.

Results
Retroviral labeling of DGCs
Retroviruses infect dividing neural progenitors and their progeny(Tashiro et al., 2006). To characterize the development of connections, we microinfused a retrovirus expressing GFP into the
DG of 42-d-old mice and conducted morphological analyses 2, 4,
6, or 8 weeks after infection (Stone et al., 2011). To contrast theconnectivity of adult-generated DGCs with DGCs born at an
earlier developmental stage, an additional group of mice was microinfused at P10, and morphological analyses were conductedRestivo et al. • Integration of Adult-Generated Hippocampal Neurons

J. Neurosci., July 22, 2015 • 35(29):10600 –10612 • 10603

terneurons (Fig. 2C; Ruediger et al.,2011). Furthermore, we found that Bassoon, a protein that is localized in active
zones of presynaptic nerve terminals (tomDieck et al., 1998), was expressed within
the filopodial-like extensions (Fig. 2D).
This is consistent with previous electron
microscopy observations that LMTassociated filopodia in CA3 contain active
zones (Acsády et al., 1998; Ruediger et al.,2011).
Development of excitatory connections
with CA3 pyramidal cells
To characterize the development of presumed excitatory contacts onto CA3 pyramidal cells, we quantified both LMT sizeand density in the CA3-sl region (Fig. 3A).
The cross-sectional area of LMTs increased as a function of post-infection delay (F(4,25) ⫽ 85.63; p ⬍ 0.0001), and, by
6 – 8 weeks after infection, GFP ⫹ LMTs
had equivalent cross-sectional areas compared with those from DGCs generated
postnatally [least significant difference(LSD) post hoc tests, p values ⬎0.05; Fig.
3B]. We observed a similar pattern ofchanges in LMT density. Density increased as a function of post-infection delay (F(4,20) ⫽ 4.70; p ⬍ 0.01), and, by 6 – 8
weeks after infection, the density of GFP ⫹LMTs was equivalent to those from DGCs
Figure 1. Retroviral labeling of newly generated DGCs. A, Scheme indicating timelines for retroviral injections and when born postnatally (LSD post hoc tests, p ⬎the animals were killed. B, Horizontal sections through the hippocampus in mice infected with the GFP-expressing retro- 0.05; Fig. 3C). These changes in LMT size
virus (green) at P10 and P42, respectively. Scale bar, 200 ␮m. Labeled DGCs extend their GFP ⫹ mossy fiber axons (inset) and density match previous reports
to the CA3-sl field. Scale bar, 50 ␮m. C, Quantification of the number of GFP ⫹ DGCs in mice microinjected at P10 (Dev) and (Faulkner et al., 2008; Toni et al., 2008;
P42 (2 w, 4 w, 6 w, 8 w). D, Percentage of GFP ⫹ DGCs coexpressing the neuronal marker NeuN in mice microinjected at P10
Sun et al., 2013) and suggest that matura(Dev) and P42 (2 w, 4 w, 6 w, 8 w).
tion of excitatory connections takes several weeks. Because larger LMTs usually
12 weeks later (Fig. 1A). After these infusions, many GFP ⫹ DGCs
contain more active zones (Pierce and Milner, 2001; Rollenhagenwere identified in the DG, and GFP-labeled mossy fibers were
et al., 2007), this suggests that, as newly generated DGCs mature,observed coursing toward the CA3 region (Fig. 1B). Consistentthey provide increasingly more powerful, high-fidelity input
with age-dependent reductions in proliferation rates, more
onto CA3 pyramidal cells (for review, see Rollenhagen and
GFP ⫹ DGCs were observed after infection at P10 compared withLübke, 2010).
P42 (F(4,15) ⫽ 58.92; p ⬍ 0.0001; Fig. 1C). Furthermore, coexpression of the mature neuronal marker NeuN increased as a
Development of excitatory connections with CA3
function of neuron age, as expected (F(4,23) ⫽ 320.3; p ⬍ 0.0001;
interneurons
Fig. 1D).
To characterize the development of output connections with local inhibitory circuits in the hippocampus, we quantified changes
Formation of excitatory connections with CA3 pyramidal
in filopodia number on LMTs in CA3-sl and en passant boutons
cells and interneurons
both in CA3-sl and the hilus. The number of filopodia/LMTs
GFP was robustly expressed after retroviral infection, allowing
depended on the post-infection delay (F(4,25) ⫽ 29.03; p ⬍for detailed analysis of mossy fiber projections as they coursed
0.0001; Fig. 4A–C). In particular, there was a pronounced spike inthrough the hilus to CA3. As reported previously, three distinct
the number of GFP ⫹ filopodia/LMTs 4 weeks after infection
types of mossy fiber presynaptic specializations were observed:(LSD post hoc tests: 4 weeks ⬎ 2, 6, and 8 weeks, p values ⬍0.05).
(1) en passant boutons in the hilus and CA3-sl that innervate only
However, by 6 – 8 weeks after infection, filopodia density deinhibitory interneurons (Acsády et al., 1998; Fig. 2 A, B); (2)clined to levels observed in developmentally labeled cells (LSD
LMTs that make excitatory contacts on spiny excrescences ofpost hoc tests, p values ⬎0.05). This transient peak was also obCA3 pyramidal neurons (Acsády and Kali, 2007; Fig. 2B); and (3)served when we estimated the total number (F(4,25) ⫽ 25.66; p ⬍filopodial-like extensions from LMTs that exclusively innervate
0.0001; Fig. 4C, inset), rather than density, of filopodia (i.e., corinhibitory interneurons in CA3-sl (Acsády et al., 1998; Lawrence
recting for post-infection changes in LMT density; Fig. 3C). Aland McBain, 2003; Fig. 2B). Counterstaining the sections forthough filopodia length was similar at all post-infection delays
parvalbumin confirmed that filopodia contact GABAergic in-10604 • J. Neurosci., July 22, 2015 • 35(29):10600 –10612

Restivo et al. • Integration of Adult-Generated Hippocampal Neurons

Figure 3. Maturation of connections with excitatory neurons. A, High-resolution images of
LMTs associated with developmentally and adult-generated DGCs (2, 4, 6, and 8 weeks after
infection). Scale bar, 5 ␮m. LMT cross-sectional area (B) and density (number of LMTs per 100
␮m; C) increases as a function of post-infection delay.

4 weeks after infection (F(4,14) ⫽ 5.72; p ⬍ 0.01; LSD post hoc tests:
4 weeks ⬎ 2 weeks, 6 weeks and 8 weeks, ps ⬍ 0.05), with levels
declining by 6 – 8 weeks post-infection to those observed in developmentally labeled cells (LSD post hoc tests; ps ⬎ 0.05). The
size of en passant boutons in CA3-sl also varied as a function of
post-infection delay with peak cross-sectional area at 4 weeks
post-infection (F4,12 ⫽ 10.75; p ⬍ 0.001; LSD post hoc tests: 4
weeks ⬎ 2, 6, and 8 weeks, p values ⬍0.05; Fig. 4F ).
In contrast, en passant bouton density in the hilus did not varywith post-infection delay [but was generally lower comparedwith those observed in developmentally labeled cells (F(4,15) ⫽3.15; p ⬍ 0.05; Fig. 4G)]. Similarly, en passant bouton area in thehilus did not vary with post-infection delay and did not differ
from those observed in developmentally labeled cells (F(4,23) ⫽0.27; p ⬎ 0.05; Fig. 4H ). This latter observation suggests that thetransient peak in connectivity to local inhibitory circuits is limited to the CA3-sl region and does not occur in the hilus.

Figure 2. High-resolution images of the three major mossy fiber presynaptic specializations.
A, Left, Retrovirally labeled mossy fiber in the hilus. En passant boutons (arrowheads) form
contacts with dendrites of inhibitory cells within the hilus. Scale bar, 5 ␮m. Right, Putative
location of dendrites of inhibitory cells within the hilus is shown as blue dotted lines. B, Retrovirally labeled cells extend their mossy fiber axon to the CA3-sl region and develop specialized
presynaptic contacts with principal excitatory CA3 pyramidal cells via LMTs (asterisk) or local
inhibitory CA3 interneurons via en passant boutons (arrowheads) or filopodia extensions from
LMTs (arrow). Scale bar, 5 ␮m. Putative locations of dendrites/thorny excrescence of principal
excitatory CA3 pyramidal cells (black dotted line) and inhibitory CA3 interneurons (blue dottedline) are depicted in the right. C, Representative GFP ⫹ filopodia (green) extending from an LMT
making contact with a parvalbumin ⫹ dendrite (gray) of an inhibitory CA3 neuron. Scale bar, 5
␮m. D, Representative GFP ⫹ LMTs and associated filopodia (green) 4 and 6 weeks after infection. Bassoon (red) is expressed in LMTs, the head of filopodia (single arrowhead), and en
passant boutons (double arrowhead). The Bassoon channel was masked using the GFP channel.
Scale bar, 2 ␮m.

(F(4,24) ⫽ 1.60; p ⫽ 0.21; Fig. 4D, inset), head size varied with
post-infection delay (F(4,18) ⫽ 11.59; p ⬍ 0.001; Fig. 4D).
A comparable pattern of changes in density of en passant boutons was observed in CA3-sl (Fig. 4E). Similar to filopodia, there
was a sharp increase in the number of GFP ⫹ en passant boutons

Development of effective connectivity with CA3 interneurons
Our results indicate that, as adult-generated DGCs integrate into
the hippocampus, they form anatomical connections with local
inhibitory circuits via transient increases in the number of filopodia extending from LMTs and en passant boutons in CA3-sl.
These results predict that stimulation of these adult-generated
DGCs should activate CA3 inhibitory interneurons and that activation should be maximal when adult-generated DGCs are 4
weeks old. To address this question, we next microinjected retrovirus expressing the light-activated cation channel ChIEF unilaterally into the DG of adult mice (Lin et al., 2009; Gu et al., 2012).
Then, at 4, 6, or 8 weeks later, we illuminated these cells via an
optical fiber implanted above the DG while mice were in theirhome cage (Fig. 5 A, B). The number of ChIEF-infected cells was
similar across groups (F(2,17) ⫽ 0.29; p ⬎ 0.05), and illuminationinduced similar Fos expression across groups (F(2,17) ⫽ 2.04; p ⬎0.05; Fig. 5C). To assess the influence of light stimulation on theactivity of different populations of inhibitory and excitatory cells
in the DG, hilus, and CA3, we examined expression of theactivity-regulated gene c-fos (Fig. 5D–F ).
The pattern of Fos expression depended on the post-infection
delay. Four weeks after retroviral infection, light stimulation ro-Restivo et al. • Integration of Adult-Generated Hippocampal Neurons

J. Neurosci., July 22, 2015 • 35(29):10600 –10612 • 10605

bustly increased Fos expression in inhibitory (GAD67 ⫹) cells in the CA3 but not in
the DG or hilus (Fig. 5G–I ). In contrast, at
6 – 8 weeks after infection, the opposite
pattern was observed. Light stimulation
increased Fos expression in GAD67 ⫹ cells
in the DG and hilus but not in CA3 (Fig.
5G–I ). A three-way ANOVA with cell age,side, and region as factors supported these
conclusions. Most importantly, this analysis revealed a three-way interaction between these factors (F(2,34) ⫽ 6.33; p ⬍0.001), and post hoc tests indicated that
artificial stimulation of 4-week-old adultgenerated DGCs strongly activated CA3
inhibitory interneurons (4 weekON ⬎ 4
weekOFF; p ⬍ 0.05), and artificial stimulation of 6- to 8-week-old adult-generated
DGCs activated inhibitory interneurons
in DG (6 weekON ⬎ 6 weekOFF and 8
weekON ⬎ 8 weekOFF; p values ⬍ 0.05) andhilus (6 weekON ⬎ 6 weekOFF and 8
weekON ⬎ 8 weekOFF; p values ⬍0.05),
respectively.
In the same mice, we found that lightstimulation of 6- to 8-week-old adultgenerated DGCs elevated overall Fos levels in presumed excitatory cells (i.e.,
GAD67 ⫺) in CA3. In contrast, light stimulation of 4-week-old adult-generated
DGCs did not affect overall Fos levels inany hippocampal subregion (Fig. 5J–L). A
three-way ANOVA with cell age, side, andregion as factors supported these conclusions. We found a main effect of side
(F(1,17) ⫽ 11.39; p ⬍ 0.001), indicatingthat Fos expression was generally elevatedin the ON versus OFF condition. This
analysis also revealed a significant threeway interaction between these factors(F(2,34) ⫽ 3.53; p ⬍ 0.05), and post hoc tests
indicated that artificial stimulation of 6to 8-week-old adult-generated DGCs
activated presumed excitatory neurons
in CA3 (6 weekON ⬎ 6 weekOFF and 8
weekON ⬎ 8 weekOFF; p values ⬍0.05).
Some limitations of this approach areworthwhile noting. Optical stimulation
only activates small numbers of adultgenerated DGCs (because of incomplete
retroviral infection and limits of lightspread). Therefore, when populationlevel changes in Fos expression are observed after stimulation, this suggests that
activity in the target population is modu4
Figure 4. Maturation of connections with inhibitory neurons. A, Representative images of LMTs associated with developmentally and adult-generated DGCs (2, 4, 6, and 8 weeks after infection). Scale bar, 5 ␮m. B, Three-dimensional reconstructions of
LMTs (orange) show the emergence of multiple filopodia (blue) from their surface. Four-week-old LMTs show the highest numberof filopodia. Scale bar, 5 ␮m. C, The number of filopodia/LMTs varies as a function of post-infection delay. Inset, Number offilopodia normalized with respect to LMT density. D, Filopodia head size varies as a function of post-infection delay. Inset, Filopodia

length does not change as a function of post-infection delay. E,
En passant density in CA3 varies as a function of post-infection
delay. F, En passant area in CA3 varies as a function of postinfection delay. G, En passant density in the hilus as a functionof post-infection delay. H, En passant area in hilus does notchange as a function of post-infection delay.

10606 • J. Neurosci., July 22, 2015 • 35(29):10600 –10612

Restivo et al. • Integration of Adult-Generated Hippocampal Neurons

Figure 5. Optical stimulation in 4-week-old adult-generated DGCs activates CA3 inhibitory interneurons. A, ChIEF– dTomato-expressing retrovirus was microinjected unilaterally into the DG in
adult WT mice. B, Four to 8 weeks after retroviral infection, the mice received a unilateral intrahippocampal optical stimulation. C, Left, Number of ChIEF-infected DGCs per slice. Right, Number of
ChIEF-infected DGCs expressing Fos after optical stimulation. Representative images of Fos expression (green) in presumed excitatory (GAD67 ⫺) and inhibitory (GAD67 ⫹) cells after lightstimulation of ChIEF (red)-infected DGCs 4 (D), 6 (E), and 8 (F) weeks after infection. Low-power scale bar, 100 ␮m; high-power scale bar, 50 ␮m. Percentage of GAD67 ⫹ cells expressing Fos after
light stimulation 4 – 8 weeks after infection in DG (G), hilus (H), and CA3 (I). Number of GAD67 ⫺ cells expressing Fos after light stimulation 4 – 8 weeks after infection in DG (J), hilus (K), and CA3(L). LSD post hoc tests, * p ⬍ 0.05 compared with corresponding OFF condition.

lated reliably by activation of adult-generated DGCs. However,
the absence of population-level changes in Fos expression does
not necessarily imply the converse. Given the small numbers of
DGCs targeted, population-level changes may fall below detectable levels. In this case, electrophysiological single-cell methods
or calcium imaging-based approaches may be more sensitive.

Additionally, there was a strong (but nonsignificant) trend forlight stimulation to induce greater levels of Fos expression inyounger cells (see Fig. 7C). This is consistent with previous observations that younger cells are more excitable and might account for the observed group differences in interneuron
activation. However, this is unlikely for two reasons. First, al-Restivo et al. • Integration of Adult-Generated Hippocampal Neurons

J. Neurosci., July 22, 2015 • 35(29):10600 –10612 • 10607

region ANOVA: main effect of cell type,F(1,44) ⫽ 33.92; p ⬍ 0.0001; main effect ofregion, F(1,44) ⫽ 44.38; p ⬍ 0.0001; andcell type ⫻ region interaction, F(1,44) ⫽13.17; p ⬍ 0.0005; Fig. 6C). In particular,
training induced an ⬃10-fold increase in
Fos ⫹/GAD67 ⫹ cells in the CA3 region(LSD post hoc tests for CA3INHIB ⬎ DGINHIB, CA3EXCIT, DGEXCIT, p values ⬍0.05).
Functional connectivity between adultgenerated DGCs and CA3 interneurons
during learning
The above results indicate that CA3 inhibitory interneurons are activated robustly
during learning and that adult-generated
DGCs transiently form strong anatomicaland effective connections with this population of cells as they integrate into adult hippocampal circuits. Together, these results
predict that these two populations of cells
should interact during hippocampusdependent learning. Therefore, using
activity-dependent gene expression to identify recently active populations of neurons,
we asked to what extent is activity coupled inthese subpopulations during learning thatengages the hippocampus (i.e., to what extent are they functionally connected; Friston
et al., 1993; Pawela et al., 2008). Because increased anatomical and effective connectiv⫹
ity with local inhibitory circuits peaked at
Figure 6. Contextual fear conditioning induces Fos in excitatory and inhibitory cells in the hippocampus. A, The number of Fos
cells in DG and CA3 after contextual fear conditioning (training) versus home cage (control). B, The percentage of GAD67 ⫹ cells ⬃4 weeks, we predicted that any changes in
expressing Fos in DG and CA3 after contextual fear conditioning (training) versus home cage (control). C, The fold increase in Fos functional connectivity would follow a simexpression in inhibitory and excitatory cells in DG and CA3 after conditioning (vs control).
ilar temporal profile.
To label large numbers of adultthough Fos expression in 4- and 8-week-old cells differed margenerated DGCs, we used a cre/LoxP-based genetic approach inginally after light stimulation (t(11) ⫽ 2.04; p ⫽ 0.061), there wasthese experiments. Specifically, mice expressing a TAM-inducible
no difference in Fos expression in 4- versus 6-week-old cells (t(11)cre recombinase driven by a progenitor-specific promoter (nestin–⫽ 1.24; p ⫽ 0.20). However, despite this lack of difference in
Cre ERT2 mice; Imayoshi et al., 2008; Arruda-Carvalho et al., 2011)activation, we found dissociable patterns of interneuron activawere crossed with YFP reporter mice (Lagace et al., 2007). In adulttion after light stimulation. That is, whereas stimulation ofoffspring from this cross, TAM treatment leads to the permanent
4-week-old cells induced Fos expression in interneurons in CA3
expression of YFP in progenitor cells and their progeny. Accordbut not hilus or DG, stimulation of 6-week-old cells produced the
ingly, P42 mice were treated with TAM and then, 4 or 6 weeks later,
opposite pattern: Fos was induced in interneurons in the hilus
were trained in contextual fear conditioning (Fig. 7A). Ninety minutes after training, brains were removed and Fos expression wasand DG but not in CA3. Second, activation of CA3 interneurons
quantified in adult-generated DGCs (i.e., YFP ⫹ cells), as were difappears to be more sensitive to stimulation frequency than total
ferent excitatory and inhibitory neuronal populations in the DG,
number of fibers activated (Mori et al., 2004).
hilus, and CA3 regions (Fig. 7B). Additional groups of reporter mice
were treated identically but not trained (i.e., age-matched homeActivation of CA3 interneurons during learning
cage controls). Although the genetic approach used here efficiently
We next examined activation of different populations of inhibilabels large numbers of adult-generated DGCs, temporal resolution
tory and excitatory cells after learning. To do this, we trained
is poorer compared with retroviral labeling strategies. Accordingly,
adult mice in contextual fear conditioning, a form of learning
we first quantified the proportion of YFP cells expressing NeuN 4
that engages the hippocampus (Kim and Fanselow, 1992), andquantified Fos expression. As expected, we found that training
and 6 weeks after TAM. YFP ⫹ cells were likelier to coexpress themature neuronal marker NeuN 6 weeks (67.37 ⫾ 2.83%) comparedelevated Fos expression (compared with home cage controls) inwith 4 weeks (50.99 ⫾ 4.08%) after TAM (t(13) ⫽ 3.24; p ⬍ 0.01),
excitatory cells in DG and CA3 (condition ⫻ region ANOVA:
suggesting that this labeled population contains a higher proportion
main effect of condition, F(1,39) ⫽ 53.90; p ⬍ 0.0001; Fig. 6A).
Similarly, we found that training elevated Fos expression in inof mature adult-generated DGCs, as predicted.
hibitory cells in DG and CA3 (condition ⫻ region ANOVA: main
To assess functional connections among different hippocameffect of condition, F(1,39) ⫽ 10.14; p ⬍ 0.005; Fig. 6B). Strikingly,
pal subpopulations, we evaluated to what degree Fos expression
the relative increase in Fos expression was greater in inhibitory
covaried in trained and control mice. Functional connectivity
compared with excitatory cells in the hippocampus (cell type ⫻may be inferred from within-subject variance or between-subject

10608 • J. Neurosci., July 22, 2015 • 35(29):10600 –10612

Restivo et al. • Integration of Adult-Generated Hippocampal Neurons

Figure 7. Correlated Fos expression in adult-generated DGCs and inhibitory interneurons in CA3 after learning. A, nestin– cre ERT2 ⫻ RosaYFP mice received 5 d of TAM injections to induce
expression of the reporter YFP in newly born neurons and were trained in contextual fear conditioning 4 or 6 weeks later. Ninety minutes after training, brains were removed and Fos immunohistochemistry was conducted. B, Fos expression was measured in seven distinct hippocampal cell populations (adult-generated DGCs and inhibitory and excitatory cells in DG, hilus, and CA3). Scale bar,
100 ␮m. Adult-generated DGCs were defined as YFP ⫹. Inhibitory cells were defined as GAD67 ⫹ and excitatory cells as GAD67 ⫺. Examples of DG–YFP ⫹, DG–GAD67 ⫹, hilus–GAD67 ⫹, andCA3–GAD67 ⫹ cells are shown in the four bottom right panels. Scale bar, 10 ␮m. C, Scatter plots showing Fos expression in 4-week-old adult-generated DGCs versus excitatory and inhibitory cell
populations in DG, hilus, and CA3. Fos expression in 4-week-old adult-generated DGCs was correlated with Fos expression in GAD67 ⫹ cells in the CA3. Fos in cell subpopulations is normalized withrespect to total hippocampal Fos for each mouse and expressed as a z-score. D, Scatter plots showing Fos expression in 6-week-old adult-generated DGCs versus excitatory and inhibitory cell
populations in DG, hilus, and CA3. Fos expression in 6-week-old adult-generated DGCs was correlated with Fos expression in GAD67 ⫺ cells in the DG and anticorrelated with Fos expression in
GAD67 ⫺ cells in the CA3 (although this correlation does not remain significant after permutation testing). E, Scatter plots showing Fos expression in developmentally generated DGCs (i.e., GAD67 ⫺cells in DG) versus excitatory and inhibitory cell populations in DG, hilus, and CA3. Fos expression in developmentally generated DGCs was anticorrelated correlated with Fos expression in GAD67 ⫹cells in DG and GAD67 ⫺ cells in hilus and CA3. F, Summaries of functional connections (inferred from correlated patterns of Fos expression across mice) for 4-week-old (4w YFP ⫹) and 6-week-old(6w YFP ⫹) adult-generated DGCs and for developmentally generated DGCs (Dev). Significant positive correlations are shown in red. Significant negative correlations are shown in blue. The dashedblue line indicates a negative correlation that did not survive permutation testing. Nonsignificant correlations are shown in gray.

variance (Mcintosh and Gonzalez-Lima, 1998). Because activity
is inferred postmortem by changes in Fos expression (yielding
single data point/mouse), here we computed covariance acrossmice to define functional connections between different neuro-nal subpopulations (Wheeler et al., 2013). After fear conditioning, we found that Fos expression in 4-week-old adult-generated
DGCs was correlated reliably with Fos expression in inhibitory(GAD67 ⫹) interneurons in the CA3 region [r ⫽ 0.66; p ⫽ 0.01

Restivo et al. • Integration of Adult-Generated Hippocampal Neurons(after permutation testing)] but not with inhibitory interneurons
in the DG [r ⫽ 0.58; p ⫽ 0.13 (after permutation testing)] or hilus(r ⫽ ⫺0.43; p ⬎ 0.05) regions (Fig. 7C). In contrast, Fos expression in 4-week-old adult-generated DGCs was not correlatedwith Fos expression in GAD67 ⫺ cells (i.e., presumed excitatory
cell populations) in the DG (r ⫽ ⫺0.45; p ⬎ 0.05), hilus (r ⫽ 0.40;
p ⬎ 0.05), or CA3 (r ⫽ 0.13; p ⬎ 0.05) regions (Fig. 7C).
We observed a different pattern for 6-week-old adultgenerated DGCs. Fos expression in 6-week-old adult-generated
DGCs was not correlated with Fos expression in inhibitory interneuron populations in the DG (r ⫽ ⫺0.13; p ⬎ 0.05), hilus(r ⫽ 0.26; p ⬎ 0.05), or CA3 (r ⫽ ⫺0.54; p ⬎ 0.05) regions (Fig.
7D). However, Fos expression in 6-week-old adult-generated
DGCs was correlated reliably with Fos expression in developmentally generated granule cells in the DG [r ⫽ 0.63; p ⬍ 0.05
(after permutation testing)]. Furthermore, Fos expression in
6-week-old adult-generated DGCs was anti-correlated with
GAD67 ⫺ cells (i.e., presumed excitatory cells) in CA3 (r ⫽⫺0.72; p ⬍ 0.05). However, this negative correlation was no longer reliable after permutation testing ( p ⫽ 0.085; Fig. 7D).
Fos expression in developmentally generated DGCs (i.e.,
GAD67 ⫺ cells in the DG granule cell layer) was correlated with
Fos expression in inhibitory interneuron populations in the DG(r ⫽ ⫺0.44; p ⬎ 0.05) but not in the hilus (r ⫽ 0.09; p ⬎ 0.05) or
CA3 (r ⫽ ⫺0.30; p ⬎ 0.05) regions (Fig. 7E). Fos expression indevelopmentally generated DGCs was anti-correlated with
GAD67 ⫺ cells (i.e., presumed excitatory cells) in the hilus (r ⫽⫺0.57; p ⬍ 0.05) and in CA3 (r ⫽ ⫺0.90; p ⬍ 0.0001; Fig. 7E).
No correlations were significant for the control groups of mice(data not shown): 4 week control mice, ⫺0.34 ⬍ r ⬍ 0.30 [all pvalues ⬎0.05 (before permutation testing)]; 6 week control mice,⫺0.51 ⬍ r ⬍ 0.57 [all p values ⬎0.05 (before permutation
testing)].
These results indicate that changes in functional connectivity
faithfully track changes in anatomical connectivity between
adult-generated DGCs and inhibitory interneuron populations.
First, the strongest anatomical and functional connections wereobserved between 4-week-old adult-generated DGCs and inhibitory interneurons in CA3. Second, similar to anatomical connections, strengthened functional connections between 4-week-old
adult-generated DGCs and inhibitory interneurons were mostpronounced in the CA3 region (Fig. 7F ). Third, the activity of
6-week-old (but not 4-week-old) adult-generated DGCs wascoupled with developmentally generated DGCs (Fig. 7F ). Finally,
the activity of 6-week-old DGCs (and developmentally generated
DGCs) was correlated negatively with the activity of CA3 excitatory cells, a finding that is consistent with previous studies showing that activation of DGCs results in net inhibition of CA3
pyramidal cell firing (Bragin et al., 1995a,b; Penttonen et al.,1997). These contrasting patterns of anatomical and functional
connectivity suggest that, as adult-generated DGCs mature, they
begin to resemble their developmentally generated neighbors.

Discussion
Here we performed a series of experiments to characterize how
adult-generated DGCs form connections with excitatory and inhibitory neurons as they integrate into hippocampal circuits.
There are three primary results. First, using viral vector approaches to label newborn neurons, we found that, as newly generated DGCs integrate into hippocampal circuits, they form
strong anatomical connections with CA3 interneurons via en
passant boutons and filopodia-like extensions from LMTs. The
number and density of these connections peaked when adult-

J. Neurosci., July 22, 2015 • 35(29):10600 –10612 • 10609

generated DGCs were 4 weeks of age, when these cells begin to
exhibit elevated excitability (Ge et al., 2007; Marín-Burgin et al.,2012). Second, optical stimulation of 4-week-old adult-generated neurons robustly activated CA3 interneurons, suggestingstrong effective connectivity between these two populations ofcells. Third, CA3 interneurons were activated strongly after contextual fear conditioning, and this activity was correlated with theactivity of 4-week-old adult-generated neurons, suggesting thatthese two subpopulations interact during learning. Although
here we focused on the formation of output connections onto
inhibitory interneurons in CA3, two recent studies (Bergami etal., 2015; Temprana et al., 2015) have identified complementarymechanisms that contribute to maintaining excitatory/inhibitorybalance as adult-generated DGCs integrate into hippocampal circuits. First, as adult-generated DGCs mature beyond 4 weeks,
they form connections with inhibitory interneurons in the DG
and hilus that provide feedback inhibition onto DGCs (Temprana et al., 2015). Second, under environmental enrichment
conditions, adult-generated DGCs are innervated transiently byinhibitory interneurons in CA1 and CA3 (Bergami et al., 2015).
Each DGC sends a single mossy fiber that projects through thehilus toward CA3 and contacts excitatory and inhibitory cells via
distinct presynaptic specializations (Acsády et al., 1998). Although these presynaptic specializations develop similarly during
development and during adulthood, the rate of maturation ismuch slower during adulthood (Overstreet-Wadiche et al., 2006;
Faulkner et al., 2008; Toni et al., 2008). For instance, in our study,
LMTs associated with adult-generated neurons appeared to be
fully mature (in terms of size) by ⬃6 weeks of age. In contrast,
during development, LMTs appear to be fully mature by ⬃4weeks of age (i.e., the beginning of postnatal week 3; Faulkner etal., 2008). Similarly, whereas we found a sharp peak in number offilopodia at ⬃4 weeks of age in adult-generated neurons, thispeak occurs earlier (at ⬃2 weeks of age) during development(Wilke et al., 2013).
The different methods we used here allowed us to describe thedevelopment of three different types of connectivity (Feldt et al.,2011). First, viral tracing revealed the development of anatomical
connectivity (i.e., the physical connections between newly generated neurons and CA3 interneurons). Second, optogenetic stimulation combined with immediate early gene expression analysis
revealed the development of effective connectivity (i.e., opticalstimulations of newborn cells activated CA3 interneurons).
Third, behavioral and immediate early gene expression analyses
revealed the development of functional connectivity (i.e., the activity of adult-generated neurons and CA3 interneurons werecorrelated during learning). Although there is not always direct
correspondence between these different forms of connectivity[Sporns, 2014; e.g., functional connections may be mediated byindirect anatomical connections (Honey et al., 2009)], here it isnotable that they appear to emerge in parallel. That is, the number and density of presumed presynaptic connections onto inhibitory interneurons in CA3 peaked when adult-generated neurons
reached ⬃4 weeks of age, and this time point corresponded withthe age at which (1) optical stimulation of adult-generated DGCs
most strongly activated CA3 interneurons and (2) activity ofthese two populations was most strongly correlated during
learning.
However, patterns of connectivity were strikingly different in
4- versus 6- to 8-week-old adult-generated neurons. In particular, our analyses revealed strong interactions between adultgenerated cells and inhibitory interneurons in CA3 at 4 weeks ofage. This pattern of strengthened connectivity with CA3 inhibi-

Restivo et al. • Integration of Adult-Generated Hippocampal Neurons

10610 • J. Neurosci., July 22, 2015 • 35(29):10600 –10612

tory microcircuits corresponds to a stage when new DGCs begin
to display elevated intrinsic excitability, reduced GABAergic inhibition, and reduced threshold for spiking and activitydependent synaptic potentiation (Schmidt-Hieber et al., 2004;
Espósito et al., 2005; Ge et al., 2007; Marín-Burgin et al., 2012).
Because CA3 interneurons provide feedforward inhibition onto
pyramidal CA3 cells (Lawrence and McBain, 2003), heightened
connectivity with CA3 inhibitory microcircuits likely provides amechanism for buffering additional excitatory drive from newborn cells.
In contrast, optical stimulation of 6- to 8-week-old adultgenerated DGCs did not induce Fos expression in CA3 inhibitory
interneurons above baseline levels. A similar pattern emerged
from the anatomical and functional connectivity analyses. By 6
weeks of age, the number and density of presumed contacts onto
CA3 inhibitory interneurons resembled those observed in developmentally generated DGCs. Similarly, during learning, wefound no correlation between Fos expression in 6-week-old
adult-generated DGCs and CA3 interneurons, suggesting thatthey were not coactive. Instead, we found that the activity of
6-week-old adult-generated and developmentally generated
DGCs were correlated strongly, suggesting that, by 6 weeks of age,
adult-generated DGCs are acquiring a more mature cellular phenotype. This conclusion is consistent with several previous investigations of the morphological and physiological properties ofadult-generated DGCs. For instance, as adult-generated DGCs
mature, they exhibit similar intrinsic excitability, spiking threshold, and activity-dependent synaptic potentiation compared with
developmentally generated DGCs (Laplagne et al., 2006; Ge et al.,2007; Mongiat et al., 2009; Gu et al., 2012). Although opticalstimulation of 6- to 8-week-old adult-generated DGCs did not
induce Fos expression in CA3 inhibitory interneurons, interestingly, we observed robust Fos expression in hilar and DG
GAD67 ⫹ cells. This raises the intriguing possibility that feedforward inhibition (via activation of CA3 inhibitory interneurons)
emerges sooner than feedback inhibition (via activation of inhibitory interneurons in the hilus and DG; Temprana et al., 2015).
Previous studies have identified maturation-dependentchanges in the morphology and physiology of adult-generated
DGCs (Espósito et al., 2005; Overstreet-Wadiche et al., 2006;
Piatti et al., 2006; Zhao et al., 2006; Ge et al., 2007; Faulkner et al.,2008; Zhao et al., 2008; Mongiat et al., 2009; Gu et al., 2012; Kimet al., 2012; Marín-Burgin et al., 2012; Dieni et al., 2013; Sun et al.,2013). The current findings add to this pattern and additionallyreveal that 4-week-old adult-generated DGCs have, at least partially, dissociable patterns of connectivity compared with moremature adult-generated DGCs. Therefore, these data underscorethe idea that adult-generated neurons represent a heterogeneouspool of cells, with different aged cohorts of cells likely contributing to hippocampal information processing in distinct ways. In
particular, we found that 4-week-old adult-generated DGCs interact strongly with CA3 inhibitory cells (and this interaction
weakens by 6 weeks of age). Previous studies suggest that DGC–
CA3 inhibitory cell connections play critical roles in memory
processing. For example, learning is associated with growth of
new filopodial synapses, and the density of these connections
correlates with memory precision (e.g., discrimination between
similar contexts; Ruediger et al., 2011). These findings suggested
that feedforward inhibition may be important for memory precision, and, consistent with this idea, genetic interventions thatprevent filopodia growth (and therefore impair feedforward inhibition) are associated with memory imprecision and impaired

population coding of similar contexts in CA3 (Ruediger et al.,2011).
Although the study by Ruediger et al. did not distinguish between adult-generated and mature DGCs, there is evidence that
adult-generated DGCs play a similar role in memory precision.
For instance, suppression of adult neurogenesis similarly impairs
memory precision, assayed by contextual discrimination paradigms (Sahay et al., 2011; Kheirbek et al., 2012; Niibori et al.,2012; Tronel et al., 2012). In one study (Niibori et al., 2012),
neurogenesis was suppressed genetically or pharmacologicallyfor 4 weeks. The loss of cohorts of immature cells (i.e., 4 weeks old
and younger) additionally impaired CA3 population coding ofsimilar contexts (assessed using cellular compartment analysis of
temporal activity by fluorescence in situ hybridization; Guzowski
et al., 2005). This impaired population coding was reflected by anincrease in the number of CA3 cells activated by two similar
contexts. However, the increase in overlap did not appear to be
driven by decreased segregation of cell populations responding toeach of the contexts. Rather, increased overlap was associatedwith an expansion of the pool of cells activated by either context.
This latter observation is consistent with the idea that the loss ofimmature adult-generated neurons impairs memory processing
by reducing feedforward inhibition. According to this model, thisloss of feedforward inhibition would decrease the activationthreshold for CA3 cells, potentially leading to less efficient encoding and/or retrieval by reducing the fidelity of CA3 population
codes. The finding that optogenetic inhibition of 4-week-old (butnot 2- or 8-week-old) adult generated DGCs impairs retrieval of
spatial memory (Gu et al., 2012) is consistent with this model.

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