Conditional reduction of adult neurogenesis impairs bidirectional hippocampal synaptic plasticity.
ABSTRACT Adult neurogenesis is a process by which the brain produces new neurons once development has ceased. Adult hippocampal neurogenesis has been linked to the relational processing of spatial information, a role attributed to the contribution of newborn neurons to long-term potentiation (LTP). However, whether newborn neurons also influence long-term depression (LTD), and how synaptic transmission and plasticity are affected as they incorporate their network, remain to be determined. To address these issues, we took advantage of a genetic model in which a majority of adult-born neurons can be selectively ablated in the dentate gyrus (DG) and, most importantly, in which neurogenesis can be restored on demand. Using electrophysiological recordings, we show that selective reduction of adult-born neurons impairs synaptic transmission at medial perforant pathway synapses onto DG granule cells. Furthermore, LTP and LTD are largely compromised at these synapses, probably as a result of an increased induction threshold. Whereas the deficits in synaptic transmission and plasticity are completely rescued by restoring neurogenesis, these synapses regain their ability to express LTP much faster than their ability to express LTD. These results demonstrate that both LTP and LTD are influenced by adult neurogenesis. They also indicate that as newborn neurons integrate their network, the ability to express bidirectional synaptic plasticity is largely improved at these synapses. These findings establish that adult neurogenesis is an important process for synaptic transmission and bidirectional plasticity in the DG, accounting for its role in efficiently integrating novel incoming information and in forming new memories.
- SourceAvailable from: Rong-Qiao He[Show abstract] [Hide abstract]
ABSTRACT: New neurons are continuously generated from resident pools of neural stem and precursor cells (NSPCs) in the adult brain. There are multiple pathways through which adult neurogenesis is regulated, and here we review the role of the N-methyl-D-aspartate receptor (NMDAR) in regulating the proliferation of NSPCs in the adult hippocampus. Hippocampal-dependent learning tasks, enriched environments, running, and activity-dependent synaptic plasticity, all potently up-regulate hippocampal NSPC proliferation. We first consider the requirement of the NMDAR in activity-dependent synaptic plasticity, and the role the induction of synaptic plasticity has in regulating NSPCs and newborn neurons. We address how specific NMDAR agonists and antagonists modulate proliferation, both in vivo and in vitro, and then review the evidence supporting the hypothesis that NMDARs are present on NSPCs. We believe it is important to understand the mechanisms underlying the activation of adult neurogenesis, given the potential that endogenous stem cell populations have for repopulating the hippocampus with functional new neurons. In conditions such as age-related memory decline, neurodegeneration and psychiatric disease, mature neurons are lost or become defective; as such, stimulating adult neurogenesis may provide a therapeutic strategy to overcome these conditions.Science China. Life sciences 04/2014; 57(4):403-11. · 2.02 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: NEW NEURONS ARE CONTINUOUSLY GENERATED IN TWO ADULT BRAIN REGIONS: the subgranular zone of the hippocampus and the subependyma by the lateral ventricles, referred to as the neurogenic niches. During their development from neural stem cells to mature functionally integrated neurons numerous choices are made, such as proliferation or quiescence, cell survival or death, migration or establishment, growth or retraction of processes, synaptic assembly or pruning, or tuning of synaptic transmission. The process is altered by physiological stimuli as well as several brain diseases. Microglia are located within the neurogenic niches and have become interesting candidates for modulating neurogenesis in both the healthy and injured brain. They become activated by foreign antigens or changes in the brain homeostasis and transform this innate immunity into an adaptive immune response by recruiting systemic immune cells. Most studies report an acute decrease in the survival of new neurons following this classically activated microglia reaction. The long-term effects are more complex. In neurodegenerative diseases, microglial activation is more heterogeneous and the transformation from a pro- to an anti-inflammatory cytokine profile and the deactivation of microglia is not well defined. The diversity is reflected by numerous reports describing both beneficial and detrimental effects on neurogenesis, primarily on the proliferation, survival, and cell fate. However, relatively few studies have investigated alterations at later stages of neurogenesis including the functional integration. Though likely, it is not established how a fine-tuned cross-talk between microglia and adult-born neurons would work and how it changes upon microglia activation. This review will therefore launch three hypotheses for how microglia might direct synaptic integration of newborn neurons, currently a fast expanding research field.Frontiers in Pharmacology 01/2012; 3:41.
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ABSTRACT: Alzheimer's disease (AD) is multifactorial and, to date, no single cause of the sporadic form of this disease, which accounts for over 99% of the cases, has been established. In AD brain, protein phosphatase-2A (PP2A) activity is known to be compromised due to the cleavage and translocation of its potent endogenous inhibitor, I2PP2A, from the neuronal nucleus to the cytoplasm. Here, we show that adeno-associated virus vector-induced expression of the N-terminal I2NTF and C-terminal I2CTF halves of I2PP2A , also called SET, in brain reproduced key features of AD in Wistar rats. The I2NTF-CTF rats showed a decrease in brain PP2A activity, abnormal hyperphosphorylation and aggregation of tau, a loss of neuronal plasticity and impairment in spatial reference and working memories. To test whether early pharmacologic intervention with a neurotrophic molecule could rescue neurodegeneration and behavioral deficits, 2.5-month-old I2NTF-CTF rats and control littermates were treated for 40 days with Peptide 6, an 11-mer peptide corresponding to an active region of the ciliary neurotrophic factor. Peripheral administration of Peptide 6 rescued neurodegeneration and cognitive deficit in I2NTF-CTF animals by increasing dentate gyrus neurogenesis and mRNA level of brain derived neurotrophic factor. Moreover, Peptide 6-treated I2NTF-CTF rats showed a significant increase in dendritic and synaptic density as reflected by increased expression of synapsin I, synaptophysin and MAP2, especially in the pyramidal neurons of CA1 and CA3 of the hippocampus.Acta Neuropathologica 11/2011; 123(1):133-51. · 9.73 Impact Factor
Conditional reduction of adult neurogenesis impairs
bidirectional hippocampal synaptic plasticity
Federico Massaa,b, Muriel Koelhb,c, Theresa Wiesnerb,d, Noelle Grosjeanb,c, Jean-Michel Revestb,e, Pier-Vincenzo Piazzab,e,
Djoher Nora Abrousb,c,1,2, and Stéphane H. R. Olieta,b,1,2
aInstitut National de la Santé et de la Recherche Médicale U862, Neurocentre Magendie, Glia-Neuron Interactions Group, F33077 Bordeaux, France;
bUniversité de Bordeaux, F33077 Bordeaux, France;cInstitut National de la Santé et de la Recherche Médicale U862, Neurocentre Magendie, Neurogenesis and
Pathophysiology Group, F33077 Bordeaux, France;dInstitut National de la Santé et de la Recherche Médicale U862, Neurocentre Magendie,
Endocannabinoids and Neuroadaptation Group, F33077 Bordeaux, France; andeInstitut National de la Santé et de la Recherche Médicale U862, Neurocentre
Magendie, Pathophysiology of Addiction Group, F33077 Bordeaux, France
Edited by Roger A. Nicoll, University of California, San Francisco, CA, and approved March 14, 2011 (received for review November 15, 2010)
Adult neurogenesis is a process by which the brain produces new
neurons once development has ceased. Adult hippocampal neuro-
genesis has been linked to the relational processing of spatial
information, a role attributed to the contribution of newborn
neurons to long-term potentiation (LTP). However, whether new-
born neurons also influence long-term depression (LTD), and how
synaptic transmission and plasticity are affected as they incorpo-
rate their network, remain to be determined. To address these
adult-born neurons can be selectively ablated in the dentate gyrus
(DG) and, most importantly, in which neurogenesis can be restored
on demand. Using electrophysiological recordings, we show that
selective reduction of adult-born neurons impairs synaptic trans-
mission at medial perforant pathway synapses onto DG granule
cells. Furthermore, LTP and LTD are largely compromised at these
synapses, probably as a result of an increased induction threshold.
Whereas the deficits in synaptic transmission and plasticity are
completely rescued by restoring neurogenesis, these synapses
regain their ability to express LTP much faster than their ability
to express LTD. These results demonstrate that both LTP and LTD
are influenced by adult neurogenesis. They also indicate that as
newborn neurons integrate their network, the ability to express
bidirectional synaptic plasticity is largely improved at these synap-
ses. These findings establish that adult neurogenesis is an impor-
tant process for synaptic transmission and bidirectional plasticity in
the DG, accounting for its role in efficiently integrating novel in-
coming information and in forming new memories.
memory|learning|glutamatergic transmission|synaptic strength
ceased (1–3). The dentate gyrus (DG) of the hippocampus is one
of the few regions of the adult mammalian brain in which
thousands of newborn cells are generated daily. The functional
role of newborn hippocampal neurons has been the subject of
extensive research and debate. Based on several correlative
pieces of evidence, it was initially hypothesized that these cells
are involved in processing spatial memory (3–5). Recently, their
participation in complex forms of hippocampal-mediated mem-
ory that require flexible, inferential memory expression has been
Because spatial learning has been associated with possible
persistent changes in synaptic strength, studies have focused
on the impact of adult neurogenesis on long-term potentiation
for glutamatergic synapses impinging on young granule cells than
for those contacting mature neurons in the DG (10). Further-
more, LTP in newborn neurons is unaffected by GABAergic in-
hibition, whereas this manipulation greatly enhances potentiation
in mature neurons (11). Our understanding of the contribution of
adult neurogenesis to synaptic transmission and plasticity in the
DG is far from complete, however. In particular, we still do not
dult neurogenesis is a process by which the brain produces
new neurons once fetal and early postnatal development has
know whether long-term depression (LTD), the inverse correlate
of LTP, is also influenced by newborn cells. Similarly, how the
plasticproperties ofthe DGnetworkare modifiedasneo-neurons
integrate their network is completely unknown.
To address these issues, we took advantage of an inducible
transgenic mouse that allows the reduction of adult hippocampal
neurogenesis (6, 12, 13). In this model, neural precursors (ex-
pressing nestin) can be selectively killed in double-transgenic mice
(bigenicmice) through overexpression of the proapoptotic protein
Bax after the oral administration of an exogenous tetracycline an-
alog, doxycycline (Dox; SI Materials and Methods). We have pre-
the processing of spatial and emotional information (6, 12, 13).
Here, using this model, we report that adult neurogenesis
influences not only LTP, but also LTD, in the DG. We further
demonstrate that as newborn neurons are incorporated in the DG
network, they improve the plastic properties of this structure by
facilitating the expression of LTP and LTD in a sequential time-
Reduction of Adult Neurogenesis Impairs Synaptic Transmission in the
DG. We first assessed whether excitatory transmission in the DG
was modified as a result of reduced adult neurogenesis in bigenic
mice treated with Dox (Bi-Dox). To this end, we recorded field
excitatory postsynaptic potentials (fEPSPs) evoked in the DG in
response to the stimulation of the medial perforant path (MPP),
one of the main excitatory inputs to granule cells. The input-
output (I/O) relationship was largely reduced in slices obtained
Bax, WT) treated with Dox (Co-Dox; Fig. 1A). This reduction did
not result from a change in the likelihood of glutamate release at
MPP terminals, because the paired-pulse ratio was not statisti-
cally different from that measured in the control animals (Fig.
1B). The most likely explanation for this change is that the re-
duction of adult-born neurons eliminated a significant number of
the MPP synapses that were participating in the evoked response
in control mice, resulting in fEPSPs of reduced amplitude.
Author contributions: F.M., M.K., J.-M.R., P.-V.P., D.N.A., and S.H.R.O. designed research;
F.M., M.K., T.W., and N.G. performed research; F.M., M.K., and D.N.A. analyzed data; and
F.M., D.N.A., and S.H.R.O. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1D.N.A. and S.H.R.O. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or Nora.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
www.pnas.org/cgi/doi/10.1073/pnas.1016928108PNAS Early Edition
| 1 of 6
Reduction of Adult Neurogenesis Alters Synaptic Plasticity. The DG
was the first structure in which persistent activity-dependent
changes in synaptic strength were documented (14). MPP synap-
sesdisplay different forms of synaptic plasticity, termed long-term
potentiation (LTP) (15) and long-term depression (LTD) (16).
We investigated the extent to which such forms of DG synaptic
plasticity depend on adult-born neurons by monitoring LTP and
LTD in both Bi-Dox and Co-Dox mice. LTP was induced by ap-
plying two trains of high-frequency stimuli (HFS; 500 ms, 100 Hz,
20 s apart). In slices obtained from Co-Dox animals, this protocol
consistently yielded a slowly developing increase in synaptic
strength that reached steady-state 15–20 min after induction (Fig.
2A). Interestingly, LTP could not be induced in Bi-Dox mice (Fig.
2A), but it was observed after GABAergic transmission was
inhibited (Fig. 2B). These data indicate that reducing the number
of adult-born neurons alters the ability to express LTP in the DG
by increasing the threshold for LTP induction, confirming pre-
vious data obtained at the level of single cell recordings (9, 10).
We next assessed the contribution of newborn neurons to LTD.
To this end, we stimulated the MPP with a low-frequency stim-
ulation (LFS) protocol (1Hz,10min).This induced areliable and
persistent decrease in synaptic strength in slices obtained from
position of the recording (R) and stimulating (S) electrodes and representative traces illustrating fEPSPs obtained in response to increasing stimulus intensity
at MPP-DG synapses in both Co-Dox and Bi-Dox mice. (Right) I/O relationship for Co-Dox mice (open circles; n = 13) and Bi-Dox mice (solid circles; n = 14). **P <
0.01, two-way ANOVA. (B) (Upper) Representative traces illustrating PPR in both Co-Dox and Bi-Dox groups. (Lower) Summary plot of PPR measured at 50-ms,
150-ms, and 300-ms interpulse intervals for Co-Dox (open circles; n = 10) and Bi-Dox (solid circles; n = 8) littermates. P > 0.05, t test.
Reduction of adult neurogenesis dramatically disrupts DG synaptic transmission. (A) (Left) Schematic representation of hippocampal circuits and
time course of fEPSP in response to HFS for Co-Dox (open circles; n = 10) and Bi-Dox (solid circles; n = 8) mice. (Upper) Representative traces obtained from Co-
Dox and Bi-Dox mice ore HFS (1) and 30 min after HFS (2). (B) Histogram summarizing LTP amplitude at 30 min after HFS in Co-Dox (open bar, 149.8 ± 14.6%;
n = 10) and Bi-Dox (solid bar, 113.9 ± 6.3%; n = 8) mice under control conditions and in Co-Dox (open bar, 172.6 ± 17.3%; n = 4) and Bi-Dox (solid bar, 137.9 ±
11.0%; n = 7) mice in the presence of bicuculline. *P < 0.05, t test. (C and D) Impaired LTD in Bi-Dox mice. (C) (Upper) Representative superimposed traces
obtaid before (1) and after (2) LTD induction. (Lower) Time course of fEPSPs after application of a LFS protocol to slices obtained from Co-Dox (open circles;
n = 10) and Bi-Dox (solid circles; n = 10) mice. (D) Histogram summarizing LTD amplitude at 30–40 min after LFS in Co-Dox (open bar, 89.3 ± 2.7%; n = 10) and
Bi-Dox (solid bar, 101.6 ± 9.8%; n = 10) mice under control conditions and in Co-Dox (open bar, 63.0 ± 8.5%; n = 9) and Bi-Dox (solid bar, 79.4 ± 3.5%; n = 9)
mice in the presence of bicuculline. **P < 0.01, t test.
Adult-born neurons are necessary for DG synaptic plasticity. (A and B) Neurogenesis blockade impairs LTP. (A) (Lower) Summary plot representing the
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| www.pnas.org/cgi/doi/10.1073/pnas.1016928108Massa et al.
Co-Dox mice (Fig. 2 C and D). Interestingly, the same protocol
failed to induce LTD in Bi-Dox mice. As for LTP, LTD could be
induced when GABAergic transmission was inhibited (Fig. 2D),
in agreement with an increased threshold for induction. Taken
together, our data indicate that adult neurogenesis is an impor-
tant contributor to the plastic properties of MPP synapses.
Different Time Courses for LTP and LTD Recovery After Adult
Neurogenesis Rescue. If the deficits that we observed in synaptic
transmission and plasticity are the direct consequence of disrupt-
ing adult neurogenesis, then they should be abrogated in the Bi-
cessation of Dox treatment. This approach also provides a unique
of the DG as these cells become incorporated in the hippocampal
We first verified that cell genesis returned to normal values
after the cessation of Dox treatment. After 4 wk of Dox impreg-
nation, Bi-Dox and Co-Dox mice were treated with the sole ve-
hicle for 2 wk. At this time point, the ability to generate newborn
cells was completely recovered (Fig. 3A). Indeed, the number of
newborn cells labeled with 5-chloro-2′-deoxychloridine (CldU) at
2 h before sacrifice was similar in Co-Dox/No-Dox and Bi-Dox/
In a subsequent experiment, we examined cell survival. Dox
was removed for one subgroup of mice from each genotype for
4 wk, after which the animals were injected with CldU and then
killed 3 wk later (Fig. 3 A and B). Dox treatment decreased the
number of 3-wk-old CldU-immunoreactive (IR) cells in bigenic
mice, and this effect was abolished when Dox was removed from
the drinking solution. To phenotype the newborn cells, we quan-
tified the percentage of CldU-IR cells expressing the neuronal
marker NeuN (Fig. 3D). Neuronal differentiation was similar
among the groups, indicating that neurogenesis in Bi-Dox mice
returned to normal values after the cessation of Dox treatment
We next analyzed whether synaptic transmission and plasticity
recover as neurogenesis returns to normal values. After 4 wk of
Dox treatment, bigenic mice were treated with the sole vehicle for
2 or 4 wk (Bi-Dox/No-Dox mice) and compared with control
animals (Co-Dox/No-Dox) that received the same protocol. At
2 wk after cessation of Dox, the I/O relationship at MPP synapses
in Bi-Dox/No-Dox mice was no longer different from that mea-
sured in control animals (Co-Dox/No-Dox; Fig. 4A). Surprisingly,
whereas LTP was perfectly normal in these animals (Fig. 4 B and
C), LTD remained significantly impaired (Fig. 4 D and E). These
animals’ ability to display LTD was not lost, as demonstrated by
the findings that at the MPP synapses that were first potentiated,
subsequent application of an LTD-induction protocol caused
depotentiation (Fig. 4 F and G). These findings suggest that 2 wk
after the cessation of Dox treatment, MPP synapses were maxi-
mally depressed, thereby preventing further LTD.
At 4 wk after cessation of Dox treatment, the I/O relationship
at MPP synapses in slices from Bi-Dox/No-Dox mice in which
neurogenesis was restored (Fig. 3) no longer differed from that
measured in Co-Dox/No-Dox animals (Fig. 5A). Similarly, both
LTP (Fig. 5 B and C) and LTD (Fig. 5 D and E) were perfectly
normal in these animals compared with control mice. Taken
together, these findings confirm that the deficits in synaptic
plasticity observed in Bi-Dox mice were caused by the disruption
of adult neurogenesis (Fig. 6).
Our conclusions are further supported by recordings obtained
from the CA1 region of the hippocampus, a nonneurogenic area.
We found that neither the I/O relationship nor LTP at Schaffer
collateral synapses were affected in Bi-Dox mice, indicating that
ablation of adult-born neurons in the DG did not affect synaptic
In this study, we tackle the importance of adult neurogenesis
for hippocampal synaptic plasticity using an inducible reversible
transgenic mouse model. This approach has several advantages,
because it is not associated with any of the side effects that ac-
company the use of irradiation or the administration of phar-
macologic compounds (17, 18). Moreover, it targets specifically
hippocampal neurogenesis without affecting olfactory neuro-
genesis (6, 12, 13). More importantly, such a partial ablation of
adult-born neurons is reversible, enabling validation of a direct
role of adult neurogenesis in governing synaptic strength in the
DG. Our data unambiguously demonstrate that reducing neuro-
genesis dramatically impairs synaptic transmission and both LTP
and LTD at MPP synapses. Previous studies using x-irradiation
demonstrated that ablation of adult-born hippocampal neurons
decreases LTP (11, 19, 20). In those studies, however, the I/O
relationship was not affected, suggesting that irradiation pro-
duced misleading results and/or that some compensatory mech-
and differentiation. All mice were first treated for 4 wk with Dox (black
boxes in the experimental design). Then Dox was removed (white boxes),
and the mice were treated with the sole vehicle for 2 wk (A) or 4 wk (B and
D). The syringes represent CldU injections. (A) At 2 wk after Dox removal, cell
genesis returned to control values (Co-Dox, n = 5; Bi-Dox, n = 6; t9= 1.369; P =
0.20). (B) Dox was removed for one subgroup of animals from each genotype
for 4 wk (Co-Dox, n = 10; Bi-Dox, n = 9). Dox treatment decreased the
number of 3-wk-old CldU-IR cells in bigenic mice (t18= 2.34; P = 0.03), an
effect abolished when Dox was removed from the drinking solution (t17=
−0.35; P = 0.72). (C) Illustration of CldU-IR cells in a Co-Dox mouse, a Bi-Dox
mouse, and a Bi-Dox/No-Dox mouse. (Scale bar: 50 μm.) (D) Cell differenti-
ation measured by the percentage of CldU-IR cells expressing the mature
neuronal marker NeuN was similar among the groups. (Right) A CldU-IR cell
(red stain) expressing NeuN (green stain).
Effect of Dox treatment and Dox removal on cell genesis, survival,
Massa et al.PNAS Early Edition
| 3 of 6
anisms accounted for this apparent discrepancy in the findings.
The simplest explanation for the diminished I/O relationship
observed in bigenic mice is the reduction in the number of syn-
apses recruited during MPP stimulation. As newborn neurons are
lost, the synapses impinging on these cells can no longer partici-
pate in the evoked-synaptic responses.
in which GABAergic transmission is left intact, whereas they can
be induced in the presence of GABA-A receptor antagonists.
These results are in agreement with previous findings reporting
newborn neurons compared with those contacting mature DG
neurons (9, 10). These findings support the involvement of adult-
born neurons in LTD. These results are not surprising when one
considers the similarity of LTP and LTD. Both depend on Ca2+
influx through NMDA receptors and lead either to an insertion
or an endocytosis of AMPA receptors (21). Thus, a very likely
explanation for the deficiency of LTP and LTD in bigenic mice is
a positive shift of the threshold for inducing synaptic plasticity.
Thanks to the use of our bigenic model, we were able to rescue
adult neurogenesis by stopping Dox treatment and monitoring
synaptic transmission and plasticity at different time windows.
Surprisingly, we found that the I/O relationship and LTP, but not
LTD, were completely restored 2 wk after the rescue of neuro-
genic functions. Because we were able to depotentiate MPP syn-
apses that were first potentiated, it appears that the absence of
LTD under these conditions is due to a floor effect in which
synapses are maximally depressed and can no longer respond to
an LTD-induction protocol. These findings suggest that newborn
neurons that are incorporated into the DG network are first
contacted by synapses that can be strengthened only through
LTP. This situation changes later as those synapses acquire the
ability to display bidirectional plasticity, as indicated by the data
obtained at 4 wk after cessation of Dox treatment.
Previous work established that synapses on newborn neurons
have an increased capacity for potentiation compared with syn-
apses impinging on mature neurons (10). This is in agreement
with our present results showing that as newborn cells are in-
corporated, the plastic properties of the network improve dra-
matically, as demonstrated by the ability of MPP synapses to first
express LTP before being able to display both LTP and LTD. It
has been proposed that these unique characteristics may make
new neurons within this critical period more sensitive to life ex-
perience. In particular, it has been shown that learning or expe-
riencing an enriched environment determines the survival, den-
dritic development, and population response of the new neurons
(22–26). This exceptional plasticity of the young neurons may be
particularly useful for information processing (27, 28).
No-Dox (open squares; n = 7) and Bi-Dox/No-Dox (solid squares; n = 7) mice. P > 0.05, two-way ANOVA. (B) Time course of fEPSPs after HFS in Co-Dox/No-Dox
(open squares; n = 8) and Bi-Dox/No-Dox (solid squares; n = 9) mice. (C) Histogram summarizing LTP amplitude measured at 30–40 min after HFS in Co-Dox/No-
Dox (open bar; 127.9 ± 3.0%; n = 9) and Bi-Dox/No-Dox (solid bar; 127.7 ± 4.6%; n = 9) mice. **P < 0.01, t test. (D and E) Failure in the rescue of LTD at 2 wk
after the cessation of Dox treatment. (D) Time course of fEPSPs after application of a LFS protocol to slices obtained from Co-Dox/No-Dox (open squares; n =
10) and Bi-Dox/No-Dox (solid squares; n = 10) mice. (E) Histogram summarizing LTD amplitude at 30–40 min after LFS in Co-Dox/No-Dox (open bar; 91.1 ± 3.0%;
n = 9) and Bi-Dox/No-Dox (solid bar; 107.4 ± 4.1%; n = 6) mice. *P < 0.05, t test. (F and G) MPP synapses are depotentiated and show LTD only if they are
first potentiated. (F) Time course of fEPSPs after application of a HFS and LFS protocols to slices obtained from Bi-Dox/No-Dox mice (solid squares; n = 8). (E)
Histogram summarizing baseline, potentiation, and depotentation amplitudes in Bi-Dox/No-Dox mice (83.8 ± 5.4%, 100%, and 83.7 ± 5.2%, respectively;
n = 8). *P < 0.05, t test.
Two wk of restoration of neurogenesis is insufficient to fully rescue synaptic plasticity. (A) I/O relationship at MPP-DG synapses measured in Co-Dox/
4 of 6
| www.pnas.org/cgi/doi/10.1073/pnas.1016928108 Massa et al.
In conclusion, adult hippocampal neurogenesis is a key pro-
cess influencing the plastic properties of the DG by facili-
tating the expression of LTP and LTD in a sequential time-
dependent manner as newborn neurons are incorporated into
the hippocampal network. This structural and functional plas-
ticity may be an important contributor to the ability to form
Materials and Methods
Subjects and in Vivo Dox Treatment. Adult male transgenic mice were obtained
from the breeding of Tet-Bax with Nestin-rtTA founder mice, as described
previously (6, 12, 13). In brief, in this model, Dox administration to bigenic
mice activates the nestin-controlled transactivator (rtTA), which drives the
transcription of the proapoptotic Bax gene in the adult brain and not in
peripheral tissues. It specifically induces death of nestin-expressing cells in the
subgranular zone of the DG, leading to reduced neurogenesis.
Eight-wk-old male nestin-rtTA/Tet-Bax bigenic mice and their control lit-
termates (nestin-rtTA, Tet-Bax, and WT) were housed individually from the
beginning of the Dox treatment through the end of the experiments. Dox
was added to the drinking solution for 4 wk (2 mg/mL, 2.5% sucrose) starting
when the mice were 8 wk of age. For the “reversal” experiments, Dox was
removed from the drinking solution for 2 wk (first batch) or for 4 wk (second
batch). In this latter experiment, Dox treatment started when the mice were
HFS (2). (C) Histogram summarizing LTP amplitude measured at 30–40 min after HFS in Co-Dox/No-Dox (open bar; 126.1 ± 8.7%; n = 5) and Bi-Dox/No-Dox (solid
bar;129.2± 8.3%; n= 6)mice.*P <0.05,t test. (D andE)RescueofLTDat4wkafterthecessationofDoxtreatment. (D)TimecourseoffEPSPsafter applicationof
an LFS protocol to slices obtained from Co-Dox/No-Dox (open rhombs; n = 10) and Bi-Dox/No-Dox (solid rhombs; n = 10) mice. (E) Histogram summarizing LTD
amplitude at 30–40 min after LFS in Co-Dox/No-Dox (open bar; 79.1 ± 8.0%; n = 6) and Bi-Dox/No-Dox (solid bar; 73.9 ± 7.2%; n = 9) mice. *P < 0.05, t test.
Four wk of restoration of adult neurogenesis completely rescues DG synaptic transmission and plasticity. (A) I/O relationship at MPP-DG synapses
treatment). The same parameters are also plotted for experiments achieved in the DG in animals in which Dox treatment was stopped for 2 wk and 4 wk (Right:
2 wk and 4 wk Dox washout). Bars represent changes in percentage measured compared with control animals for I/O and compared with baseline for LTP and
LTD. *P < 0.05.
Graph summarizing the effect of blocking adult neurogenesis in bigenic mice on the I/O relationship, LTP, and LTD in the DG and CA1 areas (Left: Dox
Massa et al. PNAS Early Edition
| 5 of 6
16 wk of age. All experiments were conducted in strict compliance with
European Convention and institutional regulations.
CldU Injection. Newborn cells were labeled by the incorporation of CldU (one
daily i.p. injection of 42.7 mg/kg—the molecular equivalent of 50 mg/kg of
BrdU—for 4 d). Animals were injected at 2 wk (first batch) or 4 wk (sec-
ond batch) after the cessation of Dox treatment and were killed at 2 h (first
batch) or 3 wk (second batch) after injection to study cell proliferation (first
batch) or cell survival/phenotype (second batch).
Electrophysiology. Mice were anesthetized with isoflurane and killed by
decapitation. Brains were rapidly removed and chilled in an ice-cold, car-
bogenated (i.e., bubbled with 95% O2-5% CO2) artificial cerebrospinal fluid
(ACSF) containing the following: 125 mM NaCl, 1.25 mM NaH2PO4, 25 mM
glucose, 2.5 mM KCl, 2.5 mM CaCl2, 2 mM MgCl2, and 25 mM NaHCO3.
Transverse entorhinal/hippocampal slices (300 μm thick) were cut using
a Leica VT1200S vibratome and incubated with cutting ACSF for 30 min at
room temperature. The slices were subsequently transferred to a holding
chamber in bathing ACSF, where they were maintained for 30 min at room
temperature until experiments began. Slices were individually transferred to
a submerged chamber for recording and continuously perfused with oxy-
genated (95% O2-5% CO2) bathing medium (3–5 mL/min). All experiments
were performed at room temperature. Extracellular fEPSPs were recorded
using glass micropipettes (2–4 mOhm) filled with normal ACSF bathing
medium. Slices from the middle hippocampus were used preferentially.
Responses were evoked by stimulation (0.1 ms duration, 0–10 V amplitude)
delivered to the middle molecular layer to stimulate the MPP using the same
glass electrodes used for the recordings. The placement of electrodes in the
MPP was corroborated by observing paired-pulse depression at 150-ms
interpulse intervals. Recordings were obtained using an Axon Multiclamp
700B amplifier (Molecular Devices). Signals were filtered at 2 kHz, digitized,
sampled, and analyzed using Axon Clampfit software (Molecular Devices).
In preliminary experiments, nestin-rtTA, Tet-Bax, and WT littermates treated
with Dox and tested for I/O relationship, paired-pulse, LTP, and LTD, demon-
strated no significant differences. We used them in equal proportions in these
experiments, in which they collectively served as the control group (Co-Dox).
Immunohistochemistry and Stereological Analysis. Mice were perfused trans-
cardially with a phosphate-buffered solution of 4% paraformaldehyde. One
in 10 free-floating sections were processed in a standard immunohisto-
chemical procedure to visualize CldU using rat anti-BrdU (1/4,000; Accurate).
Immunoreactivity was visualized by the biotin-streptavidin technique (ABC
Kit; DAKO) using 3,3′-diaminobenzidine as a chromogen. The number of IR
cells was counted under a 100× microscope objective throughout the entire
septotemporal axis of the granule and subgranular layers of the DG as de-
scribed previously (6). The total number of cells was estimated using the
optical fractionator method.
Analysis ofCellular Phenotypes.Toexaminethephenotype ofCldU-IR cells,we
incubated one in 10 with a BrdU antibody (1/1,000; Accurate), which was
revealed using a CY3−anti-rat antibody (1/1,000; Jackson Laboratory). Then
sections were incubated with a mouse monoclonal anti-NeuN antibody
(1/1,000), which was visualized with Alexa Fluor 488 anti-mouse IgG (1/1000;
Jackson Laboratory). The percentage of BrdU-labeled cells expressing NeuN
was determined throughout the DG using a confocal microscope with HeNe
and Argon lasers (Leica DMR TCSSP2AOBS).
ACKNOWLEDGMENTS. We thank D. Gonzales and M. Manse for the mouse
genotyping (genotyping platform of INSERM U862), C. Dupuy for the
excellent care of the mice, and J. M. Israel and G. Marsicano for a critical
reading of the manuscript. This work was supported by grants from Institut
National de la Santé et de la Recherche Médicale, Université de Bordeaux,
Agence Nationale pour la Recherche (to D.N.A. and S.H.R.O.), Fondation
pour la Recherche Médicale (Equipe FRM, to S.H.R.O.), and Conseil Régional
d’Aquitaine (to D.N.A. and S.H.R.O.).
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