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Optical controlling reveals time-dependent roles for
adult-born dentate granule cells© 2012 Nature America, Inc. All rights reserved.

Yan Gu1, Maithe Arruda-Carvalho2,3, Jia Wang1, Stephen R Janoschka1,4, Sheena A Josselyn2,3,5,
Paul W Frankland2,3,5 & Shaoyu Ge1,4
Accumulating evidence suggests that global depletion of adult hippocampal neurogenesis influences its function and that thetiming of the depletion affects the deficits. However, the behavioral roles of adult-born neurons during their establishment ofprojections to CA3 pyramidal neurons remain largely unknown. We used a combination of retroviral and optogenetic approaches
to birth date and reversibly control a group of adult-born neurons in adult mice. Adult-born neurons formed functional synapses
on CA3 pyramidal neurons as early as 2 weeks after birth, and this projection to the CA3 area became stable by 4 weeks in age.
Newborn neurons at this age were more plastic than neurons at other stages. Notably, we found that reversibly silencing thiscohort of ~4-week-old cells after training, but not cells of other ages, substantially disrupted retrieval of hippocampal memory.
Our results identify a restricted time window for adult-born neurons essential in hippocampal memory retrieval.
The adult hippocampus continues to give rise to several thousand new
dentate granule cells each day1–3. Accumulating evidence from studies
using global perturbation or ablation of adult hippocampal neurogenesis has revealed deficits in some forms of hippocampal memoryin rodents4–9. As the morphological and physiological phenotypesof adult-born cells change markedly as they mature, they may havedistinct roles at different stages following integration into hippocampal circuits. Accordingly, although traditional manipulations of adultneurogenesis may disrupt hippocampal memory function, it is notclear whether the observed memory deficits are a result of a global
disruption of neurogenesis.
Emerging evidence has shown that surviving adult-born dentate
granule cells (DGCs) extend dendrites and receive functional input
from the existing neural circuits as early as 2 weeks after birth10–14.
In contrast with the many studies on input synapses, little is known
about the establishment of functional projection of adult-born DGCs
to the hippocampal trisynaptic circuit. Although massive effort has
been put into determining output circuit formation of adult-bornneurons, most evidence is from anatomical examinations showingthat young newborn DGCs gradually extend axonal fibers into theCA3 area. Terminals of these axons form bouton-like structures similar to those of mature granule cells 2–3 weeks after birth15–18. A recent
study found that mature newborn neurons exhibit synaptic responses
in the CA3 area16. However, the precise timing for functional output
synapse formation and maturation remains unknown.
Input (dendritic) synapses of adult-born neurons show enhanced
plasticity between 4–6 weeks after birth compared with other stages 19,
at which point they exhibit heightened intrinsic excitability andlower activation threshold13,20. This coincides with the timing when

newborn neurons are recruited into adult neural circuits mediating­behavior21–23. These findings suggest that a cohort of young adultborn neurons of similar age may form a hypersensitive unit that
preferentially responds to stimuli during hippocampal memory formation. A related question is whether output synapses, if formed andfunctional, also exhibit heightened plasticity around the same time.
Combining retroviral birth-dating and gene delivery10,11 with
optogenetic stimulation24, we examined the behavioral roles ofadult-born neurons during their output circuit development. We
found that adult-born DGCs established functional synapses with
CA3 pyramidal neurons as early as 2 weeks after birth and synaptic
responses became stable by ~4 weeks of age. Fully established output synapses of adult-born neurons exhibited enhanced plasticity at~4 weeks after birth. Notably, optogenetic silencing of a cohort of
4-week-old, but not 2- or 8-week-old, newborn neurons substantiallyaffected the retrieval of hippocampal-dependent memory following
the completion of training, suggesting that 4-week-old new neurons
have a privileged role in hippocampal memory function. These datacharacterize the development of output circuit function for adultborn DGCs, revealing a precise time window in which newborn neurons exhibit enhanced plasticity at CA3 synapses and are critical forprocessing hippocampal memories.
RESULTS
Establishment of output synapses of adult-born neurons
To examine the role of adult-born neurons in hippocampal function
during their circuit integration, we first determined the timing fornewborn neurons to establish functional projections to CA3 pyramidal neurons. We used an optogenetic method to excite a group of

1Department of Neurobiology and Behavior, SUNY at Stony Brook, Stony Brook, New York, USA. 2Program in Neurosciences and Mental Health, Hospital for Sick

Children, Toronto, Ontario, Canada. 3Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada. 4Program in Neuroscience, SUNY at Stony Brook,
Stony Brook, New York, USA. 5Department of Physiology, University of Toronto, Toronto, Ontario, Canada. Correspondence should be addressed to
S.G. (sge@notes.cc.sunysb.edu) or P.F. (paul.frankland@sickkids.ca).
Received 27 June; accepted 15 October; published online 11 November 2012; doi:10.1038/nn.3260

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adult-born DGCs simultaneously to determine the development offunctional output circuit in the CA3 area16,24. We constructed a retroviral vector expressing EGFP-tagged Channelrhodopsin 2 (ChR2EGFP), a light-sensitive channel24,25, to birth date and specifically
express ChR2-EGFP in adult-born hippocampal neurons10,11,16.
ChR2-EGFP retroviruses were microinjected into the hilus of thedentate gyrus in adult mice and acute brain sections were prepared
1–8 weeks post infection (wpi)11 (Fig. 1a and Supplementary
Fig. 1a,b; see Online Methods). As expected, ChR2-EGFP retroviruses specifically targeted neural progenitors, and these cells
gradually developed into typical DGCs (Supplementary Fig. 1c–e).
Notably, brief pulses of blue light reliably induced action potentials
in infected DGCs, but not in non-infected neighbors (Fig. 1b), indicating that this population of cells could be optically controlled. The
infected cells were not illuminated during development and we did
not observe changes in the intrinsic properties of newborn neurons
expressing ChR2 at different ages than those expressing EGFP alone(Supplementary Table 1), which is consistent with previous findings
where optogenes were expressed in other cell types24.
Next, we used whole-cell recording to examine postsynaptic currents in CA3 pyramidal neurons while optically stimulating ChR2EGFP–positive mossy fibers of newborn DGCs (Fig. 1c). Postsynaptic
activity increased with the age of infected adult-born DGCs (Fig. 1d).
At 1 wpi, a time point at which mossy fibers have not yet reached theCA3 region, no postsynaptic responses were observed following optical stimulation. However, at 2 wpi and older (a stage at which mossy
fibers have reached the CA3 region), stimulation produced ­excitatory


b

25 mV

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ChR2+ DG cellsaChR2+ MF

Figure 1 Adult-born neurons form functional
synapses on CA3 pyramidal neurons. (a) Top,
experimental timeline. Bottom, image showing
adult-born DGCs (4 wpi) and their axons(EGFP+). Scale bar represents 50 µm. DG,
dentate gyrus. (b) Light pulses (473 nm,
5 ms) elicited action potentials in ChR2-EGFP–
infected, but not neighboring (EGFP −) DGCs.
Left, image showing recorded DGCs filledwith biocytin (white) in an acute brain slice(4 wpi). A ChR2-EGFP+ newborn neuron (green
and white) is shown in i, and a non-infectedneighbor (white) is shown in ii. Scale bar
represents 50 µm. Right, light-induced action
potentials in EGFP+ (i), but not in EGFP−(ii) DGCs. (c) Optically evoked EPSCs fromadult-born DGCs. Left, image showing ChR2EGFP+ axonal terminals (green) of adult-born
DGCs (4 wpi) and a recorded CA3 pyramidal
neuron (white). Scale bar represents 25 µm.
Right, a sample optically evoked EPSCs
recorded from this cell, subsequently blocked
by 50 µM CNQX. (d) Axon integration (left,
EGFP) and formation of functional synapses
on CA3 neurons (right) at 1, 2, 3 and 4 wpi.
Scale bar represents 10 µm. (e) Amplitude of
EPSCs at 1, 2, 3, 4, 6 and 8 wpi (P < 0.05,
n = 5–15 cells, two-tailed unpaired t test).
(f) Latency of EPSCs at 2, 3, 4, 6 and 8 wpi.
(g) Summary of EPSCs that were inhibitedby 50 µM l-AP4 (t = 5.465, P = 0.012,
n = 5 cells, two-tailed paired t test) or 1 µM
LY354740 (t = 3.891, P = 0.027, n = 6 cells,
two-tailed paired t test) and blocked by 50 µM
CNQX (t = 5.835, P = 0.007, n = 15 cells,
two-tailed paired t test). All values represent
mean ± s.e.m. *P < 0.05.

20 pA© 2012 Nature America, Inc. All rights reserved.

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postsynaptic responses (EPSCs). These optically evoked EPSCs
reached maximal responses at 4 wpi (Fig. 1d,e). They had a latencyof ~4 ms and were blocked by an AMPA receptor antagonist (CNQX)
and metabotropic glutamate receptor agonists (L-AP4 and LY354740,
specifically blocking mossy fiber–CA3 synapses 26), indicating thatthey represented a glutamatergic monosynaptic response (Fig. 1f,g).
These results suggest that young adult-born DGCs form functional
synapses with CA3 target cells, which become stable around 4 wpi.
These findings, together with those of previous studies showing newborn neurons to be functionally innervated by entorhinal corticalprojections10–12, indicate that adult-born DGCs fully integrate into
the hippocampal trisynaptic circuits by ~4 wpi.
Heightened synaptic plasticity of young adult-born neurons
We next characterized functional properties of these output synapses.
We examined synaptic plasticity of young adult-born DGCs in the CA3
as previously described13,19. To optically stimulate CA3 synapses of agroup of adult-born neurons at high frequency, we replaced the ChR2
construct with a ChR2 variant, ChIEF-dTomato27, which responds
more reliably to high-frequency optical stimulation. As expected,
3-, 4- and 8-week-old adult-born DGCs expressing ChIEF responded
reliably to theta-burst optical stimulation (TBS), a procedure used forlong-term potentiation (LTP) induction13,19,27,28 (Supplementary
Fig. 2a), to their soma (Supplementary Fig. 2b,c). Recordings fromaxonal boutons revealed that, following optical stimulation, activation insoma could reliably propagate to the axonal terminals (Supplementary
Fig. 2d,e), which is consistent with previous observations in matureadvance online publication

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not mature)
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Accordingly, we next
1.050
examined whether the­activity of T-type Ca2+
0.5
channels contri­buted to00
the heightened plasticity.
After establishing reliable recordings fromthe mice at 3, 4 and 8 wpi, we blocked T-type Ca2+ channels withintraperitoneal injection of ­mibefradil (25 mg per kg of body weight),
a specific T-type Ca2+ channel blocker, and examined their role in
the expression of output synaptic plasticity30 (see Online Methods).
Mibefradil showed no effect on the basal synaptic transmission(Supplementary Fig. 5a,b). Notably, optical TBS failed to induce
an enhancement of synaptic transmission from mibefradil-treatedmice at 3 and 4 wpi (Fig. 2e), at which point we observed robust
potentiation in control animals using the same induction procedure(Fig. 2e). Although mibefradil is considered to be a specific blocker,
it has been reported to block R-type Ca2+ channels of some cultured
cells at low dose31. To exclude this possibility, we performed recordings in hippocampal slices from mice at ~4 wpi. Consistent with ourin vivo data, application of mibefradil (1 µM) abolished LTP of young
DGCs, whereas SNX482 (500 nM), an R type–specific antagonist,
failed to affect this LTP (Supplementary Fig. 5c–f). When mice weretreated with mibefradil at ~8 wpi, LTP seemed to be unaffected by themibefradil application. Because of the small LTP expression using TBSat this age, we were unable to determine the effect precisely (Fig. 2e).
However, together with previous observations of specific activation ofT-type Ca2+ channels in young neurons13, these data suggest that theactivity of T-type Ca2+ channels in young adult-born DGCs likely contri­
butes to the heightened output synaptic LTP. Combined with previous
studies on intrinsic excitability and input synapses13,19,32, these results
reveal that a cohort of fully integrated (~4 weeks old) young newborn
DGCs have more excitable membranes and enhanced input/output
synaptic plasticity.
4

implanted an optic fiber in the dentate gyrus to deliver optical stimulation and recorded field excitatory postsynaptic potentials (fEPSPs)in the CA3 region in vivo (Fig. 2a and Supplementary Fig. 3). fEPSPs were successfully induced by short pulses of optical stimulation(Fig. 2b). Similar to postsynaptic activity in vitro (Fig. 1c), fEPSPs
recorded in vivo were blocked by local application of CNQX (Fig. 2b).
As expected, high-frequency optical stimulation (~5 mW mm−2 intensity, 50 Hz, 2 s) evoked consistent fEPSPs (Fig. 2b). Together with thereliable responses to TBS recorded from the soma or axonal terminalsof newborn neurons (Supplementary Fig. 2), this result suggests thatthe experimental system is suitable for characterizing output synaptic plasticity of newborn DGCs using TBS. Thus, we next delivered
TBS, and measured fEPSPs slope before and after TBS19 (see Online
Methods). We found that optical TBS at 3 and 4 wpi induced LTP offEPSPs in the CA3 area in nearly all of the tested mice (Fig. 2c,d).
When the same induction procedure was used to induce LTP at 8 wpi,
only half of the tested mice exhibited potentiation (Fig. 2c,d). Thus,
output synapses of adult-born DGCs at 4 wpi exhibit a lower induction
threshold for LTP, similar to their input synapses13,19. We then analyzed
LTP amplitude at 3, 4 and 8 wpi and found that LTP amplitude was
maximal at 4 weeks. Relatively smaller LTP was observed in mice thatwere stimulated at 3 and 8 wpi (Fig. 2c,e). To determine whether thesubstantially reduced LTP at 8 wpi resulted from a lack of plasticity inthe output synapses of newborn neurons around this age, we used astronger induction procedure, tetanic optical stimulation (50 Hz, 2 s),
for LTP induction while recording pyramidal cells (Supplementary
Fig. 4a). Using this stronger induction protocol, we recorded sustained potentiation (Supplementary Fig. 4b,c), suggesting that theoutput synapses of mature newborn DGCs remain plastic, but have ahigher induction threshold. However, using the same tetanic opticalstimulation, we found a substantially higher level of LTP expression
by stimulating young newborn neurons compared with that induced
by stimulating mature newborn neurons (Supplementary Fig. 4b–d),consistent with our observation using theta-burst induction (Fig. 2).
These results indicate that young newborn neurons exhibit heightened
plasticity that peaks at ~4 wpi (Fig. 2e).
We next asked what might contribute to the heightened plasticity
in young newborn neurons. A recent study found that T-type Ca 2+channels facilitate dendritic synaptic plasticity in young newborn (butb Control Saline CNQX 50 Hz optical (2 s)

Optical3

DGCs29. To assess global circuit output of newborn neurons, we thena ChIEF-dTomato Recording

Normalized fEPSPs

Figure 2 Adult-born neurons at 4 weeks of age show enhanced plasticity
at output synapses. (a) Experimental timeline. (b) Optically stimulating
adult-born neurons produced fEPSPs in the CA3 area. Left, fEPSPs
were blocked by infusion of 50 µM CNQX (but not saline). Right, opticalstimulation (50-Hz pulses of 5 ms) reliably induced fEPSPs. Scale bars
represent 5 ms and 0.1 mV. (c) Theta-burst optical stimulation of adultborn neurons produced LTP at CA3 synapses in an age-dependent manner.
Top, examples of fEPSPs LTP from a single animal at 3, 4 or 8 wpi. Insets,
averaged traces of fEPSPs from five consecutive recordings before (1),immediately following (2) and after (3) LTP induction using TBS (bluearrow; Supplementary Fig. 2). Bottom, summary of LTP from groups ofanimals, respectively. (d) Percentage of mice (3, 4 and 8 wpi) exhibiting
reliable LTP. (e) Summary of the mean potentiation of fEPSPs amplitude
45–60 min following TBS from mice under control condition (3, 4 and 8 wpi
with 6, 8 and 8 animals, respectively; t test between groups: 3 versus
4 wpi, t = 3.386, P = 0.007; 8 versus 4 wpi, t = 4.483, P = 0.002)
or after application of mibefradil (25 mg per kg, intraperitoneal; t test
between control and mibefradil conditions: 3 wpi, t = 3.823, P = 0.007,
n = 4; 4 wpi, t = 5.656, P < 0.001, n = 5; 8 wpi, t = 0.954, P = 0.41,
n = 4). In each group, all animals tested with stable baseline were
included. All values represent mean ± s.e.m. n.s., not significant.
P > 0.05, *P < 0.05, two-tailed unpaired t test.

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Silencing young newborn neurons affects memory retrieval
Accumulating evidence suggests that the effectiveness of ablationsof adult neurogenesis depend on the timing of manipulation2,5,7,8,23.
This suggests that adult-born DGCs may assume distinct behaviorala r t ic l e s
a

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Swim speed (cm s )Light beam
Figure 3 Reversibly silencing 4-week-old adult-born neurons impairs
Arch-EGFP
Optic-rotatory joint
No light
50 pA
hippocampal memory retrieval. (a) Optical stimulation (589 nm) silences
OpticalLight
20 Hz, 5 minArch-EGFP expressing adult-born neurons. Scale bars represent
20
100 ms and 30 mV. (b) Schematic drawing showing a mouse with
–76 mV
implanted optrodes connected to an orange light source via optic fibers
10
HiddenVisibleand an optic rotatory joint. (c) Timeline of water maze experiment.
Training P1
Training P3P2
P4(d) Animals were trained with no light. Shown is the training curve of0
latency to find the platform. (e) Optically inactivating a cohort of
Hidden Visible
4-week-old adult-born neurons impaired hippocampal memory retrieval.
4 weeks
During the probe, mice in the no light condition spent significantly moretime searching in the target quadrant (NE) compared with the otherNo light
NW
NE
Hiddenquadrants (one-way repeated-measures ANOVA, F3,39 = 7.139, P < 0.001;
Light
40
50
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SE
NE > NW, SW, s.e.m. by paired t test planned comparison, n = 14),
*
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showing robust spatial memory. In contrast, mice in the light condition
600
30
Visible(inactivation) didn’t spend significantly more time in NE compared with
20
400
20
the other quadrants (F3,39 = 0.9655, P = 0.4187; P > 0.05 NE versus.
10
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P4
P3
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NW, SW, SE; n = 14), showing a disruption of spatial memory (NEno light
P1 P2
0
0
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> NElight, t = 2.153, P = 0.0253 by planned comparison). (f) Optical
1 2 3 4 5 6 1 2
Hidden Visible
inactivation did not alter swim speed in hidden probe tests (P1 and P2,
Training sessions
No light Lightt = 1.046, P = 0.3145, n = 14) and visible probe tests (P3 and P4,
t = 0.6464, P = 0.2646, n = 14). (g) Optical inactivation did not alterswim distance in hidden probe tests (P1 and P2, t = 1.008, P = 0.1660, n = 14) and visible probe tests (P3 and P4, t = 0.0173, P = 0.09867,
n = 14). All values represent mean ± s.e.m. *P < 0.05, one-way ANOVA or two-tailed t test.

cgPercent search

Swim distance (cm)e© 2012 Nature America, Inc. All rights reserved.

N
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Latency (s)droles as they mature and that these distinct behavioral roles may coincide with changes in their synaptic integration and plasticity (Fig. 2e).
To test this hypothesis, we used optogenetic stimulation to reversibly
silence groups of different aged adult-born neurons during behavioral
tasks. We generated a retrovirus to express an inhibitory optogene,archaerhodopsin-3 (Arch-EGFP)33. To maximize infection of adultborn neurons, we performed two retroviral injections (spaced 10 hapart) per mouse using a standard protocol11 (see Online Methods).
We successfully labeled ~1,700 newborn DGCs per mouse at 4 wpi.
Arch expression in labeled adult-born DGCs had no observable effecton the development of newborn neurons (Supplementary Table 1). In
acutely prepared brain sections, pulses of optical stimulation specifically silenced Arch-expressing adult-born DGCs, indicating that theactivity of these neurons could be reliably and reversibly inhibited bylight (Fig. 3a), as previously reported33.
To examine the role of a group of newborn neurons in hippocampal memory, we used a hidden platform version of the water
maze task34. We microinjected Arch-expressing retroviruses into thehilus of the hippocampus in adult mice and implanted customized
optrodes to ensure sufficient light delivery into the dorsal hippocampus bilaterally (see Online Methods and Supplementary Fig. 6). At
4 wpi, mice were trained in the water maze with optic fibers connected
to an orange light source via an optic-rotatory joint. Half of the mice
were trained with the light off (no light group) and half with the lighton (light group) (Fig. 3b and Supplementary Fig. 7a). Across thetraining trials, latencies to locate the hidden platform declined andthere was no difference in escape latencies between control (no light)
and inactivated (light) groups (Supplementary Fig. 7b), indicatingthat silencing this cohort of ~4-week-old adult-born DGCs does
not interfere with memory acquisition.
We then assessed the effect of silencing newborn neurons on memory retrieval. At ~4 wpi, mice were trained in the water maze with
optic fibers connected to an inactive light source (no light), and themice learned to locate the hidden platform (Fig. 3b–d). Following thecompletion of training, spatial memory was assessed in two probe
tests. During the first probe, half of the animals received light stimulation for the duration of the test (light) and half were tested without
the light (no light). On the following day, the animals were re-tested
in a second probe trial with the light conditions reversed (Fig. 3c,d).
Using this within-subject design, we found that inactivation of thiscohort of 4-week-old adult-born neurons robustly decreased the percentage of the time that the mice spent searching in the target quadrant (Fig. 3e). Notably, light illumination of 4-week-old EGFP-labelednewborn neurons did not affect the percent time in the target quadrant(Supplementary Fig. 8). Furthermore, light-induced inactivation did
not affect motor coordination, as swim speed (Fig. 3f) and distancetraveled (Fig. 3g) did not differ between the no light and light conditions. Memory impairments depended on both retroviral expressionof Arch and light illumination, as neither retroviral expression of Archalone (without illumination) nor illumination alone (in the absenceof Arch) impaired memory10 (Supplementary Fig. 8). These results
suggest that a cohort of ~4-week-old newborn cells is important for
memory expression. By examining expression of the activity-regulated
gene, c-Fos, we next asked whether this population of cells was normally activated by memory recall and whether this activation was
absent following light-induced inactivation. Mice received injections ofArch- or EGFP-expressing retrovirus into the dentate, and were trainedin the water maze task 4 weeks later. At the completion of training, halfof the mice were given a probe test with the light on and the other halfwith no light (Supplementary Fig. 9a). Using standard procedures21,
90 min after the probe tests, we killed the mice and performed c-Fos
staining. We imaged c-Fos+ and EGFP+ Arch+ DGCs (Supplementary
Fig. 9b). Mice in the Arch and no light and EGFP groups searchedselectively in the probe test, as expected. Following this probe test, wefound that about 4% of labeled adult-born DGCs were c-Fos positive(Supplementary Fig. 9c), consistent with our previous findings21. In
contrast, mice in the Arch and light group searched less selectively
in the probe test (similar to deficits observed in Fig. 3d), and very
few Arch-labeled adult-born DGCs expressed c-Fos (Supplementary
Fig. 9b,c), confirming the efficiency of optical inhibition and providing support for the conclusion that labeled ~4-week-old adult-born
DGCs regulate spatial memory retrieval.
We next evaluated whether silencing these neurons interfered withthe expression of a non–hippocampus-dependent memory. The same
mice were trained in a visible version of the water maze in which theplatform was located in the opposite quadrant of the pool and marked
by a visible cue. Across 2 d of training, mice learned to navigate to thecue from different start positions (Fig. 3c,d). During two subsequentadvance online publication

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The behavioral role for newborn DGCs relies on their age
We next asked whether silencing different aged cohorts of newborn neurons would have a similar effect on hippocampal memory
retrieval. To address this, we trained mice at 2 or 8 wpi. Silencing
cells that were ~2-week-old at the time of training did not disrupt
expression of spatial memory (Fig. 5a). Similarly, silencing cells thatwere ~8 weeks old at the time of training, an age at which they are
considered to be mature11,12,17 (Fig. 1), did not affect expression of a
spatial memory (Fig. 5b). Thus, silencing newborn neurons at ~4 wpi,
but not at 2 or 8 wpi, resulted in deficits in spatial memory retrieval(Fig. 5c). Furthermore, silencing newborn neurons at 2 or 8 wpi did
not affect contextual fear memory (Fig. 5d).
Not all adult-born DGCs survive, and the absence of effects ofsilencing at 8 weeks might be a result of there being fewer Arch+ cells.
Accordingly, we counted Arch+ newborn DGCs after behavioral tests.
We found that there was no obvious decrease in number of Archlabeled neurons from 4 to 8 wpi (4 wpi, 1,746 ± 52 labeled neurons
per animal, n = 17; 8 wpi, 1,721 ± 59 labeled neurons per animal,n = 10; P = 0.23, two-tailed unpaired t test), consistent with previous
findings1,4,36. Furthermore, we found that Arch+ cells had similar
dendritic complexity ~4 and 8 wpi (4 wpi, 9.85 ± 1.08 dendrites per
50 µm × 50 µm, n = 17; 8 wpi, 10.27 ± 0.95 dendrites per 50 µm × 50 µm,
n = 10; P = 0.78, two-tailed unpaired t test).
Finally, we asked whether the coincidently heightened plasticity(Fig. 2) contributes to the role of young newborn neurons in retrievalof hippocampal-dependent memories. Given that we found thatthe activity of T-type Ca2+ channels is essential for the heightened
plasticity of output synapses, we tested hippocampal memory of theArch-injected mice at 4 wpi with intraperitoneal administration ofmibefradil (25 mg per kg). Notably, the application of mibefradil
substantially decreased the searching time in the target quadrant

2w

probe tests, we found that light inactivation did not interfere with thelatency of the mice to find the visible platform (no light, 4.92 ± 0.26 s;
light, 4.87 ± 0.34 s; t = 0.1215, P = 0.4526, n = 14; two-tailed unpairedt test), swimming (Fig. 3f) or distance traveled (Fig. 3g).
To ask whether our results would generalize to another form ofhippocampus-dependent memory, we next trained a new group ofanimals in a fear-conditioning procedure35 (Fig. 4a). Mice weretrained with a single tone-shock pairing and tested in the same context
1 d later. In this test, optogenetic silencing reduced levels of conditioned freezing (Fig. 4b,c), indicating that inactivating 4-week-old
adult-born neurons impairs the retrieval of contextual fear memory.
The following day, we placed the mice in an alternate context and presented the tone. In contrast, optogenetically silencing these neurons
did not affect conditioned freezing of the mice to the tone (Fig. 4c).
As the retrieval of contextual, but not tone, fear memories depends on
hippocampal function35, these results suggest that silencing a cohortof 4-week-old adult-born DGCs impairs retrieval of a hippocampusdependent contextual fear memory.

Percent search© 2012 Nature America, Inc. All rights reserved.

Percent
freezing (contextual)

Contextual Tone
Figure 4 Temporary silencing of 4-week-old
No light
No light
newborn neurons impairs expression of a fear
60
40
Training
Light
Light
conditioning memory. (a) Timeline of fear30
conditioning test. Adult mice were infused with
4
40
*
weeks
a retroviral vector (Arch-EGFP), implanted with
20
optrodes and trained in fear conditioning (single
20
tone-shock pairing). Contextual fear memory was assessed
10
24 h after training. An additional tone test was performed in which mice were placed in a novel00context and the tone replayed. Animals were divided into two groups and optical silencing wasContextual Tone
1 2 3 4 5
counterbalanced in contextual and tone freezing tests. (b) Silencing of adult-born DGCs at 4 wpi
Time (min)reduced freezing to the context compared with controls (n (number of animals per group) = 7, 7).
(c) Optically inactivating adult-born DGCs at 4 wpi reduced freezing to the context (first 2 min of the context test, t = 2.239, P = 0.0224, n = 7, 7),
but had no effect on tone fear memory (t = 1.675, P = 0.0599, n = 7, 7). To avoid potential interference from within session extinction, we measured
freezing time in the first 2 min. All values represent mean ± s.e.m. *P < 0.05, two-tailed t test.

No light

Light

No light

Light

SalineMibefradil

Figure 5 Behavioral roles of adult-born DGCs are sensitive to their age. (a,b) Silencing adult-born DGCs at 2 or 8 wpi showed no significant effecton memory retrieval. Mice at 2 (a) or 8 (b) wpi spent more time searching in the target quadrant (NE) than the other quadrants in both no light (oneway repeated-measures ANOVA: 2 wpi, F3,15 = 15.25, P < 0.0001; 8 wpi, F3,15 = 8.081, P = 0.0019; NE > NW, SW, s.e.m. by paired t test planned
comparison in both; n = 6, 6) and light conditions (2 wpi, F3,15 = 9.125, P = 0.0011; 8 wpi, F3,15 = 14.20, P = 0.0001; NE > NW, SW, s.e.m. in
both; n = 6, 6). (c) Silencing adult-born DGCs affected memory retrieval in an age-dependent manner. The 4 wpi group showed significantly less time
searching in the target quadrant in the light condition (n = 14) compared with the 2 wpi (n = 6, t = 2.360, P = 0.030), 8 wpi (n = 6, t = 2.922,
P = 0.0135), and EGFP 4 wpi (n = 8, t = 2.392, P = 0.0286) groups by two-tailed unpaired t test. (d) Silencing of adult-born DGCs at 2 or 8 wpi failed
to affect fear memory retrieval. Percent freezing of Arch animals at 2 wpi (t = 0.2029, P = 0.4246, n = 5), 8 wpi (t = 0.3824, P = 0.3568, n = 8) or
EGFP at 4 wpi (t = 0.4593, P = 0.3326, n = 6). Two-tailed paired t test comparison was made between no light and light conditions. (e) Mibefradil
prevented hippocampal memory retrieval of the animals at 4 wpi. Shown is a summary similar to those in a (salineno light, F3,9 = 10.43, P = 0.0027,
NE > NW, SW, SE; salinelight, F3,9 = 2.849, P = 0.0975; mibefradilno light, F3,9 = 1.173, P = 0.3730; mibefradillight, F3,9 = 1.061, P = 0.4128;
n = 4, 4 for all). All values represent mean ± s.e.m. *P < 0.05; n is the number of animals per group, one-way ANOVA or two-tailed t test.

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advance online publicationa r t ic l e s© 2012 Nature America, Inc. All rights reserved.

compared with the saline and no light groups (Fig. 5e). In anothergroup of mice, in which we applied mibefradil and optical inhibition,
we observed a similar deficit in memory retrieval in the mibefradiltreated mice (Fig. 5e). Because the activity of T-type Ca2+ channels
is also required for the heightened plasticity in dendritic synapses13,
these data suggest that heightened plasticity in young newborn neurons may be important for memory retrieval. To further explore theage specificity of these effects in the same cohort of newborn neurons, we designed a within-subject experiment in which mice were
tested with the water maze at 4 wpi and contextual fear conditioningat 8 wpi, or vice versa (Fig. 6). We found that silencing newborn
neurons at 4 wpi, but not 8 wpi, disrupted memory retrieval in bothtasks, consistent with our observations (Figs. 3–5). Together, ourdata suggest that adult-born neurons may be transiently critical for
memory retrieval, but the effect, at least in spatial and contextual
memory retrieval, may decline as these adult-born neurons continue
to mature.
DISCUSSION
We used optogenetic methods to evaluate how adult-born DGCs functionally integrate into hippocampal circuits. We found that newborn
DGCs form functional glutamatergic synapses in the CA3 area as
early as 2 wpi and that these synapses become functionally stable by
4 wpi. The output synapses of 4-week-old newborn neurons exhibited enhanced plasticity. Notably, reversibly silencing a population of
4-week-old, but not 2- or 8-week-old, newborn DGCs affected hippo­
campal memory retrieval. These data indicate that adult-born neurons
influence hippocampal function and behaviors in a maturationdependent manner.
Functional output circuit development
We successfully employed a retroviral method to deliver optogenes
into cohorts of adult-born DGCs. As we previously reported10,11,
infection with these retroviruses did not appear to affect the development of labeled newborn neurons (Supplementary Fig. 1 andSupplementary Table 1). By using optogenetic stimulation, physio­
logy and imaging, we found that adult-born neurons functionally project to the CA3 neural circuitry. Blockade of postsynaptic
responses by applying AMPA receptor antagonists or metabotropic
glutamate receptor agonists revealed that they are typical mossy fiber
synapses26 (Fig. 1e). Together with several previous studies10–12,17,
our results suggest that adult-born DGCs fully incorporate into the
hippocampal trisynaptic neural circuit around 4 wpi.
Percent search

Percent freezing(contextual)

Percent freezing(contextual)

Percent search

Figure 6 Task-switching experiments showing
Fear at 8 wpi
Fear at 4 wpi
Water maze at 8 wpi
a Arch Water maze at 4 wpi
b Archthat adult-born DGCs at 4 wpi are importantfor memory retrieval. (a) Top, timeline of water
maze and fear-conditioning tests. Bottom, at
4 wpi, water maze–trained animals under theContextual
Contextual
no light condition searched selectively (ANOVA,
40
40
50 *
50 *
*
F3,12 = 9.487, P = 0.0017; NE > NW, SW,
40
40
30
30
SE; n = 5, 5; see Figs. 3–5), whereas in the
*
30
30
20
20
light condition they failed to search selectively
20
20(F3,12 = 4.004, P = 0.0345; NE versus NW,
10
10
10
10
t = 0.0702, P = 0.43; NE versus SW, t = 1.792,0000
P = 0.077). When trained in the contextual
t
t
t
t
E
E
E E
E
E E
E
h
h
h
h
N NWSW S N NWSW S
N NWSW S N NWSW S
ig
ig
lig
lig
fear conditioning at 8 wpi, animals displayed a
L
L
o
o
N
N
No light
Light
No lightLightsimilar percent freezing (t = 0.3600,
P = 0.3685). (b) Top, timeline of fear conditioning and water maze tests. Experiments were performed as described in a, but the order of behavioral
tests was switched: fear condition and then water maze. Bottom, animals showed a significant decrease in percent freezing (t = 3.046, P = 0.0191)
when trained at 4 wpi. However, at 8 wpi, both no light and light groups showed intact water maze memory (no light, F3,12 = 14.48, P = 0.0003;
light, F3,12 = 9.644, P = 0.0016; NE > NW, SW, SE; n = 5, 5). All values represent mean ± s.e.m. *P < 0.05, n is the number of animals per group,
one-way ANOVA or two-tailed paired t test.

We further found that output synapses of adult-born DGCs exhibited enhanced plasticity at ~4 wpi (Fig. 2). This mirrors the time during
which we observed hyper-sensitive input synapses and during whichthese cells have more excitable membranes13,19. Mechanistically, we
found that mibefradil, a T-type Ca2+ channel blocker, was able to abolish the heightened axonal plasticity of young newborn neurons, similar
the effect it has on their dendritic synapses13. Although it is possiblethat mibefradil affects R-type Ca2+ channels31, heightened plasticity
was not blocked by the application of SNX-482, an R-type Ca2+ channel
antagonist. Considering previous studies showing its specificity30,37,38
and young DGCs representing a hyperactive population with highlevel T-type Ca2+ channels13, we think that T-type Ca2+ channels arevery likely the major targets of mibefradil. In addition, the mechanisms
described in the dendritic synapses of adult-born DGCs19,20,26,39 maycontribute to the heighted axonal plasticity40. Although the precise
mechanisms remain to be determined, impaired spatial memory­following blockade of T-type channels suggest that these channels haveimportant functional roles in adult-born neurons.
Time-dependent role of young adult-born neurons
Chemical, genetic and irradiation-based methods have been widelyused to ablate neurogenesis and explore the role of adult neurogenesis
in hippocampal function5,41. Although these studies have suggested
that adult neurogenesis is important for hippocampal memory, they
are limited in that these manipulations typically affect differently aged
adult-born cells. Using retrovirally expressed optogenes, we were able
to address this issue by labeling and silencing distinct cohorts of adultborn neurons. This optogenetic silencing of adult-born neurons by
high-frequency light stimulation is likely to result from effective, but
not excessive, hyperpolarization, rather from than potentially harmfulchanges in internal chloride levels33.
Our results suggest that silencing of ~4-week-old newborn neurons
leads to memory deficits. In contrast, silencing this same population before training did not prevent the acquisition of a hippocampal
memory. The dissociation is consistent with our recent findings using
a transgenic diphtheria toxin–based ablation system to study the roleof adult-born neurons in hippocampal memory7 and suggests thatlearning may occur in the absence of newborn neurons. However, if
newborn neurons are present and functional in circuit level at the timeof training, they are recruited into hippocampal memory circuits,
and silencing (or ablating7) these cells revealed that they are essential for memory retrieval. In our previous diphtheria toxin–based
ablation study, adult-born cells spanning a range of ages (including
advance online publication

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a r t ic l e s~4-week-old cells) were targeted7. Our finding that substantial memory retrieval deficits were only observed after silencing of ~4-weekold newborn neurons raises the possibility that the deficits following
post-training ablation observed in our previous study were primarily
driven by the loss of ~4-week-old newborn neurons. Still, we cannot
exclude the possibility that silencing ~8-week-old newborn neurons
also affects hippocampal function, albeit to a lesser degree. Finally,
it is noteworthy that the retroviral approach only labeled a relatively
small group of newborn neurons, and, thus, even the activity of thissmall population of cells strongly influenced hippocampal function. Although silencing a similar population before training did not
impair memory acquisition, it is likely that silencing a larger population might induce anterograde memory deficits.
Methods
Methods and any associated references are available in the online
version of the paper.
© 2012 Nature America, Inc. All rights reserved.

Note: Supplementary information is available in the online version of the paper.
Acknowledgments
We thank J. Bischofberger, G. Matthews, L. Role and H. Song for their criticalcomments, and Q. Xiong and J. Tucciarone for technical support. We thank themembers of F. Gage’s laboratory for sharing their behavioral protocols. This
work was supported by US National Institutes of Health (NS065915), American
Heart Association (0930067N), Feldstein Medical Foundation and State Universityof New York Research Excellence in Academic Health grants to S.G. and Canadian
Institutes of Health Research grants to P.W.F. (MOP86762) and S.A.J. (MOP74650).
AUTHOR CONTRIBUTIONS
Y.G. conducted all of the electrophysiological, immunohistochemical and confocal
imaging analyses. Y.G. and M.A.-C. performed all of the behavioral analyses. J.W.
engineered retroviral constructs and Y.G. produced retrovirus. S.R.J. helped withsome initial manuscript preparation. S.G. and P.W.F. supervised the project. S.G.,
P.W.F., S.A.J., Y.G. and M.A.-C. wrote the manuscript. All of the authors read anddiscussed the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/doifinder/10.1038/nn.3260.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.

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ONLINE METHODS© 2012 Nature America, Inc. All rights reserved.

Retroviral production and stereotaxic injection. Engineered self-inactivating
murine oncoretroviruses were used to deliver genes of interest specifically to
proliferating cells and their progeny10,11. The optimized ChR2 constructs wereobtained from K. Deisseroth (Stanford University) and the ChIEF constructfrom R. Tsien (Addgene). The Arch construct from E. Boyden was purchasedfrom Addgene.
Purified engineered retroviruses were stereotaxically injected into adult
C57BL/6 mice (Charles River). 5–6-week-old female mice were used for all
experiments. For behavioral experiments, animals in each experimental groupin general had slight different birth dates to minimize the potential effects fromthe estrous cycle. All mice were housed under standard conditions with 12-h:12h dark/light cycle. All animal procedures were conducted in accordance withinstitutional guidelines and were approved by the Institutional Animal Care and
Use Committee of Stony Brook University.
Optrode implantation and behavioral procedures. Optrodes (Doric Lenses,
modified to increase light spread) were implanted bilaterally into the dorsal dentate gyrus (coordinates: 3.0 mm rostral from bregma, 2.6 mm lateral from the
midline and 2.5 mm ventral) 14 d after two retroviral injections (~10-h interval)
unless we specifically indicated otherwise in the main text. After implantation,
animals received at least 2 weeks recovery before any behavioral experiment.
After behavioral experiments, mice were perfused transcardially withphosphate-buffered saline (PBS) followed by 4% (w/v) paraformaldehyde (PFA).
Brains were sectioned and the optrode implantation sites were verified and numbers of retroviral-labeled adult-born neurons were counted. Mice were excluded if
the implantation site was incorrectly positioned. Mice with missed viral injections
were discarded. Mice with correct injections all had 1,500–2,000 newborn DGCs
and all of them were selected for behavioral analysis.
Water maze (hidden platform version). The apparatus and behavioral procedures have been described previously21,42. Behavioral testing was conducted in acircular water maze tank (120 cm in diameter, 50 cm deep), located in a dimly lit
room. The pool was filled to a depth of 40 cm with water made opaque by adding white, non-toxic paint. Water temperature was maintained at approximately
26 °C. A circular escape platform (10-cm diameter) was submerged 0.5 cm below
the water surface, in a fixed position in one of the quadrants. The pool was surrounded by curtains, at least 1 m from the perimeter of the pool. The curtains
were white with distinct cues painted on them.
Water maze training took place across 6 d. Each training session consistedof three training trials (inter-trial interval was ~15 s). On each trial, mice wereplaced into the pool, facing the wall, in one of four pseudorandomly varied startlocations. The trial was complete once the mouse found the platform or 40 s had
elapsed. If the mouse failed to find the platform on a given trial, it was guided
onto the platform by the experimenter. Following training, spatial memory wasassessed in two probe tests with the platform removed from the pool. The probe
tests were 40 s in duration and conducted 24 h and 48 h after the last training
session. Animals performed training and probes attached to the optic fibers androtatory joint. Each animal experienced one probe with light stimulation and one
without, the order of which was counterbalanced between animals. Behavioral
data from training and the probe tests were acquired and analyzed using an
automated tracking system (Ethovision XT, Noldus). Using this software, werecorded a number of parameters during training, including escape latency andswim speed. In probe tests, we measured the amount of time mice searched in
the target quadrant versus the three other quadrants.
Water maze (visible platform version). For the visible platform task, the platform
was moved to the opposite quadrant and marked by a visual cue. The cue consistedof a plastic cylinder (4 cm in diameter, 4 cm in height) with a horizontal black
and white vertical-striped pattern and was placed on top of the platform. Visible
platform training started 24 h after the last hidden platform probe and consistedof one training session of three trials per day (inter-trial interval was ~15 s) across
2 d. On each trial, mice were placed into the pool, facing the wall, in one of four startlocations (pseudorandomly varied). The trial was complete once the mouse foundthe escape platform or 40 s had elapsed. Similar to the hidden version of the watermaze, animals were divided into two groups to perform two probe tests, counterbalanced for order of light stimulation. The probes started 24 h after training and were

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conducted on consecutive days. As before, behavioral data from training and theprobe tests were acquired and analyzed using an automated tracking system.
Context fear conditioning. The fear conditioning chamber consisted of a stainlesssteel conditioning chamber (18 cm × 18 cm × 30 cm, Coulbourn) containing a
stainless steel shock grid floor. Shock grid bars (diameter = 3.2 mm) were spaced
7.9 mm apart. The grid floor was positioned over a plastic drop-pan, which waslightly cleaned with 70% ethyl alcohol to provide a background odor. The front of thechamber was made of clear acrylic and the top, back and two sides made of modular
aluminum. Animals were subjected to two probes, a context test and a tone test.
For the context testing, animals were placed in the fear chamber, where they wereoriginally shocked. For tone testing, mice were put in a modified version of the fearchamber that consisted of a white, plastic floor covering the shock grid bars and aplastic, triangular insert placed inside the same conditioning chamber used for training. One of the walls of this insert had a black- and white-striped pattern. The othertwo walls were white. After each test, the plastic floor was cleaned with water. Mousefreezing behavior was monitored via overhead cameras and scored manually.
During training, mice were placed in the fear conditioning chamber for a totalof 3 min. After 2 min of free exploration mice were presented with a 30 s tone
(2,800 Hz, 85 dB) that co-terminated with a 2-s foot shock (0.5 mA). Mice remainedin the chamber for a further 30 s before being returned to their home cage.
We assessed freezing in two 5-min tests 24 h after training, in the fear chamber
and its modified version, respectively. In the second probe, the tone was presented
after a 2 min delay. Animals were divided into two groups for counterbalanced
tests of the animals’ freezing to the context and the tone, respectively. Data ispresented as function of time for the context test. For the tone test, we measured
freezing to the tone for 60 s.
Slice and in vivo physiology. Mice were processed at 1, 2, 3 and 4 wpi and electrophysiological recordings performed at 32–34 °C, as previously described11. For
efferent CA3 synapse slice experiments, short pulses of blue light were generated
by a 50-mW, 473-nm laser under the control of a standard digital converter board
and launched into a Zeiss upright microscope through the epifluorescence lightpath. The ending power on brain slices was ~5 mW mm−2 and synaptic transmission was recorded at –65 mV.
For in vivo recording, mice received injections of ChIEF-dTomato retrovirus
3, 4 and 8 weeks before in vivo recordings. Briefly, animals were anesthetized andmounted on a stereotaxic frame. An optrode was inserted into dorsal dentate
gyrus (coordinates: 3.0 mm rostral from bregma, 2.6 mm lateral from the midline and 2.5 mm ventral). Pulses of blue light controlled by recording software(pClamp10.0) was generated from a 50-mW, 473-nm laser and delivered thought
an optic fiber. A recording electrode was inserted into CA3 molecular layer (coordinates: 2.0 mm rostral from bregma, 2.2 mm lateral from the midline and 2.2 mm
ventral), and fEPSPs were recorded following optical stimulation of the dentate
gyrus. After recording, mice were perfused transcardially with PBS followed by
4% PFA. Brains were sectioned to check for the presence of retroviral-labeled
adult-born neurons and the sites of stimulation in dentate gyrus and recording
in CA3 (Supplementary Fig. 3). Mice were excluded if the recording site wasout of CA3. Slope of fEPSPs in each trace was measured and averaged every
2 min for analysis and plotting. For each animal, fEPSP slope values in the last
15 min (45–60 min) were compared with baseline values (–20–0 min) by twotailed independent t test. Recording was considered a successful LTP if fEPSP slope
values in the last 15 min were significantly greater than baseline (P < 0.05).
c-Fos immunohistochemistry and analysis. Neuronal activity during memory
retrieval was analyzed by imaging immediate-early gene c-Fos expression aspreviously discribed24 (Supplementary Fig. 9a). Briefly, 90 min following thecompletion of behavioral testing, mice were deeply anesthetized and perfusedtranscardially with PBS and then 4% PFA. Brains were removed, fixed overnightin PFA and then transferred to a 30% (w/v) sucrose solution and stored at 4 °C.
Brains were sectioned into 50-µm coronal sections covering the full anterior-posterior extent of the dentate gyrus. Immunohistochemistry was performed using
primary antibody to c-Fos (rabbit polyclonal antibody, 1:1,000, Calbiochem,
catalog no. PC38) and Alexa 568–conjugated goat antibody to rabbit (1:500,
Molecular Probes, catalog no. A11011) as the secondary antibody. Sections wereincubated in primary antibody overnight, and then with secondary antibody for
2 h at room temperature (25 °C), in the presence of 2% goat serum, 1% bovine

doi:10.1038/nn.3260

serum albumin and 0.2% (w/v) Triton X-100. Sections were mounted on slides
with Permafluor anti-fade medium (Lipshaw Immunon). Images of the dentate
gyrus were taken on an Olympus FLV1000 confocal microscope, and we quantified c-Fos+ and EGFP+ Arch+ cells throughout the anterior-posterior extent of
the granule cell layer. The numbers of c-Fos+ cells and EGFP+ Arch+ cells were
quantified from two-dimensional images of the entire dentate gyrus, and the ratioof c-Fos+ to EGFP+ Arch+ cells was calculated for each animal.

42. Teixeira, C.M., Pomedli, S.R., Maei, H.R., Kee, N. & Frankland, P.W. Involvementof the anterior cingulate cortex in the expression of remote spatial memory.
J. Neurosci. 26, 7555–7564 (2006).

© 2012 Nature America, Inc. All rights reserved.

Staining and reconstruction of biocytin-filled neurons. Brain sections withbiocytin-filled neurons were fixed in 4% PFA overnight and stained using an
Alexa 647–conjugated streptavidin from Invitrogen. Images were acquired on
an Olympus FLV1000 confocal system and biocytin-filled neurons were reconstructed afterwards using Olympus FluoView10 software.

Statistical analysis. Data were analyzed using ANOVAs followed by t tests.
Significance was considered to be P < 0.05. ANOVAs and most t tests are describedin the figure captions. In the water maze, animals were considered to be searching selectively if the percent search in the northeast quadrant was significantly
greater than that for each of the other quadrants (that is, northeast > northwest,
northeast > southeast, and northeast > southwest). If the time spent in the northeast quadrant was not greater than that spent in all of the other quadrants, thesearch was considered to be not selective. For all behavioral experiments, animals
were randomly grouped and data analysis was blinded. Cell counting and c-Fos
analysis were performed blinded.

doi:10.1038/nn.3260

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