imaging of abGCs revealed that dendritic spines were significantly more stable in lactating mothers compared with naive virgins. In
contrast, spine stability of resident GCs remained unchanged after parturition. In addition, while spine size distribution of abGCs was
bulbar circuitry of lactating mothers. This enhanced integration may serve as a cellular mechanism, supporting changes in olfactory
Across the animal kingdom, the transition into motherhood is
accompanied by well characterized behaviors, which ensure re-
productive success. In most mammals, this form of behavioral
plasticity starts at parturition, or shortly thereafter, and is char-
acterized by intensive protection and nursing of the offspring. In
pup retrieval, pup licking and crouching (Noirot, 1969). While
the transition into motherhood has been studied extensively at
lates of these changes remain largely unknown.
In many animal species, olfaction plays a central role in
numerous basic functions; one of which is social recognition
(Schaefer et al., 2002; Brennan and Kendrick, 2006; Baum and
Kelliher, 2009; Keller et al., 2009). In the context of maternal
behavior, the olfactory system may be particularly important for
the vicinity of the nest. Indeed, in mice, olfaction is essential
for mothers to behave maternally toward their offspring
(Gandelman et al., 1971; Vandenbergh, 1973). Increasing ev-
idence in numerous species suggest that neural plasticity, al-
ready at the level of the olfactory bulb (OB), is important for
maternal recognition and for the production of normal ma-
ternal behaviors (Dickinson and Keverne, 1988; Le ´vy et al.,
1990; Kendrick et al., 1997; Sakamoto et al., 2011).
population that can serve as a good candidate for the plasticity
following parturition. In this respect, the abINs are an attractive
population because they are continuously incorporated into the
OB’s circuitry in large numbers throughout adulthood (Altman,
for structural and functional plasticity (Lledo et al., 2006;
Lazarini and Lledo, 2011). With regard to maternal care, it has
been shown that in the absence of adult neurogenesis maternal
behaviors are impaired (Sakamoto et al., 2011). Moreover adult
neurogenesis, which is enhanced during pregnancy, is followed
by a significant increase in the number of newly generated abINs
in the OB (Shingo et al., 2003). The integration process of these
neurons that were born during pregnancy is expected to occur
peak. The correlation between the increased surge of adult-born
gests that synaptic connections made by abINs are an integral
part of OB plasticity during this time.
In this study, we characterized structural plasticity of abINs
that were born during pregnancy and integrated into the OB
circuitry during the first stages of motherhood. We focused on
assistance throughout this project, S. Rumpel and Y. Loewenstein for advice on spine size analysis, I. Nelken for
TheJournalofNeuroscience,May30,2012 • 32(22):7519–7527 • 7519
adult-born granule cells (abGCs), which are the major cell pop-
ulation that undergoes neurogenesis throughout adulthood in
the OB (Lledo et al., 2006). Using in vivo time lapse two photon
imaging as well as confocal imaging, we describe the structural
synaptic properties of newly born neurons in the OB of lactating
mothers. We show that abGCs’ dendritic spines in mothers be-
a new aspect of cellular plasticity unique to this early station of
Animals. BALB/C mice (n ? 31 mice, 10–14 weeks old) were used in all
experiments. The control group included naive virgin females that were
placed in a cage with a proven breeder male. After 3 d, the male was
separated from the cage and after 7 d, the females were injected with
niche). This group consisted of lactating mothers that gave birth to at
we did not find a correlation between the mothers’ litter size (as an
indirect proxy for maternal behavior) (Seitz, 1958; Grota and Ader,
fore, all maternally behaving mothers were grouped into a single group.
All experimental procedures used in this study were approved by the
Hebrew University Animal Care and Use Committee.
Lentiviral vectors. To generate recombinant lentiviral vectors we sub-
cloned a fusion protein of Synaptophysin and GFP (kind gift from D.
In addition, to express GFP we used transfer plasmids with GFP under
the control of the CMV promoter or synapsin promoter. Virus was pro-
duced by transfection of human embryonic kidney cells (HEK293) with
third-generation lentivirus plasmids using polyethylenimine. The me-
dium was collected after 36 h and again after additional 24 h. Virus was
concentrated using ultracentrifugation and resuspended in PBS.
Stereotaxic injection of lentivirus into the stem cell niche. Lentivirus in-
jections into the subventricular zone (SVZ) and into the rostral migra-
tory stream (RMS) were performed as described earlier (Mizrahi, 2007).
midine (0.83 mg/kg). Carpofen (0.004 mg/g) was injected subcutane-
ously before surgery. One of the following lentiviruses was used to
promoter and lentivirus encoding for Synaptophysin-GFP (Syn-GFP),
driven by the EF-1-? promoter. Virus injections (0.2–0.5 ?l) were done
stereotaxically into one hemisphere, using pressure (coordinates relative
to bregma; SVZ: anterior, 1 mm; lateral, 1 mm; depth, 2.2 mm; RMS:
anterior, 3.3 mm; lateral, 0.8 mm; depth, 2.9 mm). After surgery, mice
fully recovered and were returned to the animal facility under normal
housing conditions. Both naive and pregnant females returned to their
original cages at groups of 2–3 females. Approximately 8 d later (?2 d
before parturition), mice from both groups were placed in separated
cages until imaging or perfusion.
experiment of resident GCs we used the cranial window preparation,
the skull overlying both OBs was carefully removed, leaving the dura
intact. Lentivirus-GFP driven by synapsin promoter was injected into
both bulbs to transduce resident neurons in the OB. Then, a 3 mm
and sealed in place using histoacryl (TissueSeal) and dental cement. A
0.1 g metal bar was glued to the skull for repositioning the animal’s head
under the microscope in consecutive imaging sessions (Mizrahi and
Katz, 2003). After surgery, mice fully recovered and returned to the ani-
mal facility under normal housing conditions. Approximately 8 weeks
later, mice were anesthetized and underwent the first imaging session
with no further surgical interference (see below, In vivo two-photon
In vivo two-photon imaging. Time lapse imaging of abGCs in lactating
corresponded to 14–17 d post-injection (d.p.i.) at the first imaging ses-
imaging session). Imaging was performed in anesthetized freely breath-
Livneh et al., 2009). Time lapse imaging of resident GCs was performed
through the implanted cranial window. In both experiments, the mouse
a metal bar glued to the skull in a fixed orientation relative to the objec-
tive lens. Mice were imaged using a two-photon microscope (Prairie
Technologies) equipped with a 40? (0.8 NA) IR–Achroplan water-
immersion objective (Olympus). A femtosecond laser (Mai-Tai Spectra
Physics) was used to excite GFP at 900–920 nm. Images (512 ? 512
pixels) were acquired at 0.23 ?m per pixel resolution in the XY dimen-
sion and 0.9 ?m steps in the Z dimension. Each dendritic tree was iden-
tified in the additional imaging sessions by its location in 3D relative to
the blood vessel map (Mizrahi, 2007; Livneh et al., 2009).
Histology and tissue processing. Fifteen to 18 d.p.i. (corresponding to
day 4 following parturition in the lactating mothers group), mice were
perfused transcardially with 0.9% saline followed by 4% paraformalde-
hyde and the brains were cryoprotected in 30% sucrose. OBs were sec-
tioned coronally (40 ?m) on a sliding microtome. Syp-GFP was then
amplified as described by others (Kelsch et al., 2008). Briefly, slices were
incubated overnight with a primary antibody (rabbit anti-GFP, 1:1000,
Millipore), and then with a secondary antibody (goat anti-rabbit-Cy3,
1:500, Jackson ImmunoResearch) for 3 h. Both antibodies were diluted
Confocal microscopy. Slices were imaged at 0.165–0.2 ?m/pixel in the
XY dimension and at 0.5 ?m steps in the Z dimension. Imaging was
performed with an Olympus FV-1000 confocal microscope, via a 40?
(1.25 NA) oil objective. We imaged and analyzed only distal GC den-
drites in the external plexiform layer (EPL), as different parts of the GC
dendritic tree were shown to have different properties (Kelsch et al.,
we limited our analysis to the brightest neurons in the sample. This bias
to high contrast signal may be one reason for the higher average spine
densities than what we and others have reported earlier (Whitman and
Greer, 2007; Dahlen et al., 2011; Livneh and Mizrahi, 2011; but see also
for the higher average spine densities is the way we scored spines with
multiple heads (see below, Data analysis).
Data analysis. All analyses in this study were done blind to the exper-
imental condition. Quantitative analyses of spine dynamics were per-
formed manually from the filtered image stacks (Gaussian blur filter)
sessions were aligned to each other using the “Sync windows” plugin
(http://rsb.info.nih.gov/ij/plugins/sync-windows.html). Each spine was
then scored as stable, lost, or gained using the “cell counter” plugin
(http://rsb.info.nih.gov/ij/plugins/cell-counter.html). For each cell,
spine dynamics were calculated as follows: number of stable spines ?
Nstable/(Nstable? Nlost), number of lost spines ? Nlost/(Nstable? Nlost),
number of gained spines ? Ngained/(Nstable? Ngained). Nstable, Nlost, and
Ngainedare the number of stable, lost, and gained spines, respectively.
signal that clearly contrasted from the background. To estimate the size
was performed by using custom software written in Matlab (Math-
Works). First, each frame of the stack was filtered with a 3 ? 3 pixel
median filter. Then, for each spine its head, adjacent background, and
was calculated in two dimensions at the image frame in which the inte-
grated fluorescent of a single spine head was highest. Intensity values of
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