The Journal of Neuroscience, September 3, 2014 • 34(36):11919 –11928 • 11919

Development/Plasticity/Repair

Conditional Deletion of ␣-CaMKII Impairs Integration of
Adult-Generated Granule Cells into Dentate Gyrus Circuitsand Hippocampus-Dependent Learning
X Maithe Arruda-Carvalho,1,2 Leonardo Restivo,1 Axel Guskjolen,1,3 Jonathan R. Epp,1 Ype Elgersma,4Sheena A. Josselyn,1,2,3,5 and Paul W. Frankland1,2,3,5
1Program in Neurosciences and Mental Health, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada, 2Institute of Medical Science, Universityof Toronto, Toronto, Ontario M5S 1A8, Canada, 3Department of Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada, 4Department of
Neuroscience, Erasmus MC University Medical Center, Rotterdam, The Netherlands, and 5Department of Psychology, University of Toronto, Toronto,
Ontario M5S 3G3, Canada

New granule cells are continuously integrated into hippocampal circuits throughout adulthood, and the fine-tuning of this process islikely important for efficient hippocampal function. During development, this integration process is critically regulated by the␣-calcium/calmodulin-dependent protein kinase II (␣-CaMKII), and here we ask whether this role is conserved in the adult brain. To dothis, we developed a transgenic strategy to conditionally delete ␣-CaMKII from neural progenitor cells and their progeny in adult mice.
First, we found that the selective deletion of ␣-CaMKII from newly generated dentate granule cells led to an increase in dendritic
complexity. Second, ␣-CaMKII deletion led to a reduction in number of mature synapses and cell survival. Third, consistent with alteredmorphological and synaptic development, acquisition of one-trial contextual fear conditioning was impaired after deletion of ␣-CaMKII
from newly generated dentate granule cells. Previous work in Xenopus identified ␣-CaMKII as playing a key role in the stabilization ofdendritic and synaptic structure during development. The current study indicates that ␣-CaMKII plays a plays a similar, cellautonomous role in the adult hippocampus and, in addition, reveals that the loss of ␣-CaMKII from adult-generated granule cells is
associated with impaired hippocampus-dependent learning.
Key words: CaMKII; adult neurogenesis; hippocampus

Introduction
During development, neuronal structure is dynamic. Initiallydendritic growth is rapid, with frequent addition and retractionof dendritic branches. However, as dendritic arbors mature andbecome more complex, growth slows and structure stabilizes.
Calcium/calmodulin-dependent protein kinase II (CaMKII) is
thought to play a key role in this stabilization process. For example, in the developing Xenopus, the onset of CaMKII expression
coincides with a reduction in dendritic growth rate and stabilization of dendritic arborization (Wu and Cline, 1998). Overexpres-

Received Feb. 14, 2014; revised July 21, 2014; accepted July 24, 2014.
Author contributions: M.A.-C., L.R., and P.W.F. designed research; M.A.-C., L.R., A.G., and J.R.E. performed research; Y.E. contributed unpublished reagents/analytic tools; M.A.-C., L.R., and A.G. analyzed data; M.A.-C., S.A.J.,
and P.W.F. wrote the paper.
This work was supported by Canadian Institutes of Health Research Grant MOP86762 to P.W.F. and Grant
MOP74650 to S.A.J. and Brain Canada to P.W.F., M.A.-C. was supported by fellowships from the Ontario Graduate
Scholarship program and The Hospital for Sick Children. We thank R. Kageyama and I. Imayoshi for the nestinCre ERT2 mice; and B. Richards, G. Vetere, A. de Cristofaro, R. Braybon, M. Yamamoto, and M. Sakaguchi for discussions and technical assistance.
The authors declare no competing financial interests.
Correspondence should be addressed to Dr. Paul W. Frankland, Program in Neurosciences and Mental Health,The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. E-mail:paul.frankland@sickkids.ca.
DOI:10.1523/JNEUROSCI.0652-14.2014
Copyright © 2014 the authors 0270-6474/14/3411919-10$15.00/0

sion of CaMKII prematurely initiates stabilization of dendritic
arborization by reducing rates of branch addition and retraction(Wu and Cline, 1998). Conversely, inhibition of CaMKII activity
increases neurite complexity and branch dynamics (Wu andCline, 1998; Zou and Cline, 1999). Consistent with this role indendritic and axonal maturation, CaMKII regulates synaptic
maturation (Wu et al., 1999). The role of ␣-CaMKII in activitydependent structural stabilization appears to be conserved in the
mammalian brain. For example, in the developing rodent brain,
CaMKII expression peaks postnatally (Sugiura and Yamauchi,
1992; Tang et al., 2004; Nakashiba et al., 2012), and genetic inhibition of the ␣ isoform of CaMKII increases dendritic complexityof mouse dorsal horn neurons during development (Pattinson etal., 2006). Similarly, CaMKII regulates structural stabilization inneuronal cell lines (Goshima et al., 1993; Tashima et al., 1996;
Massé and Kelly, 1997) and primary neuronal cultures (Redmond et al., 2002).
These data indicate that during development initially dynamic
neuronal structure is stabilized by ␣-CaMKII-dependent processes (see also Wang et al., 1994). Neurogenesis persists beyond
development in the subgranular zone of the hippocampus; therefore, here we test whether this role for ␣-CaMKII in stabilizationof neuronal structure is conserved when new neurons integrate
into established circuits in adult mice. To address this, we used a

Arruda-Carvalho et al. • ␣-CaMKII and Hippocampal Neurogenesis

11920 • J. Neurosci., September 3, 2014 • 34(36):11919 –11928

transgenic strategy to delete ␣-CaMKII in a Cre recombinaseinducible manner (␣-CaMKII f/f mice). To restrict ␣-CaMKII deletion to neural progenitor cells, we crossed ␣-CaMKII f/f micewith nestin-Cre ERT2 mice, in which a tamoxifen (TAM)inducible Cre recombinase is expressed under the control of anestin promoter (Imayoshi et al., 2008). In adult offspring fromthis cross, TAM administration deletes ␣-CaMKII in neural progenitor cells (NPCs) and their progeny. We found that deletion of
␣-CaMKII led to a an increase in complexity of adult-generated
granule cells in the dentate gyrus (DG), suggesting that
␣-CaMKII plays a cell-autonomous role in stabilization of dendritic structure as new neurons integrate into established hippocampal circuits. Further, we found a reduction in the numberof mature synapses onto ␣-CaMKII-deficient newly generated
granule cells, and, consistent with this, impaired learning in ahippocampus-dependent task. These results suggest that
␣-CaMKII regulates integration of adult-generated granule cells
into the dentate gyrus by arresting dendritic growth and stabilizing input connections.

Materials and Methods
Mice

Nestin-CreERT2 mice. Nestin-Cre ERT2⫹ mice express a TAM-inducible
form of Cre recombinase driven by a progenitor specific (nestin) promoter, as previously described (Imayoshi et al., 2008; Arruda-Carvalho etal., 2011). In all experiments, we used line 4 from Imayoshi et al. (2008)
as recombination efficiency is highest in the subgranular zone of the
hippocampus in this line (Imayoshi et al., 2008; Arruda-Carvalho et al.,2011).
␣-CaMKIIf/f mice. In ␣-CaMKII floxed mice, ␣-CaMKII f/f exon 2 ofthe ␣-CaMKII gene is flanked by loxP sequences (Achterberg et al.,2014). In all the experiments, homozygote mice were used, so that Cre
recombinase-mediated excision of exon 2 led to complete knock-out of
␣-CaMKII in cells in which Cre recombinase is active.
Rosa26-LacZ mice. The Rosa26-LacZ reporter mice have been previously described (Zambrowicz et al., 1997). In this reporter line, LacZ
expression is under the control of the ubiquitous Rosa26 locus promoter,and expression of the LacZ transgene is dependent on the Cre
recombinase-mediated removal of a transcriptional STOP cassette. We
crossed this reporter line with nestin-Cre ERT2 and ␣-CaMKII f/f mice tocharacterize recombination and ␣-CaMKII deletion.
All lines were maintained on a C57BL/6 background (Taconic Farms).
Genotypes were determined by PCR analysis of tail DNA samples aspreviously described (Zambrowicz et al., 1997; Buch et al., 2005; Imayoshi et al., 2008). Two breeding schemes were used. For all behavioral andmorphological analyses, nestin-Cre ERT2⫹/␣-CaMKII f/f mice were bredwith ␣-CaMKII f/f/Rosa26-LacZ ⫹/⫹ mice, resulting in nestin-Cre ERT2⫹/
␣-CaMKII f/f/Rosa26-LacZ ⫹/⫺ and nestin-Cre ERT2⫺/␣-CaMKII f/f/Rosa26-LacZ ⫹/⫺ offspring. For all remaining analyses (proliferation,
survival, maturation), nestin-Cre ERT2⫹/␣-CaMKII f/⫹ mice were bredwith ␣-CaMKII f/⫹/Rosa26-LacZ ⫹/⫹ mice, resulting in nestin-Cre ERT2⫹/
␣-CaMKII f/f/Rosa26-LacZ ⫹/⫺,
nestin-Cre ERT2⫹/␣-CaMKII f/⫹/Ro⫹/⫺
ERT2⫹
sa26-LacZ, nestin-Cre/ ␣ -CaMKII ⫹/⫹ /Rosa26-LacZ ⫹/⫺ ,nestin-Cre ERT2⫺/␣-CaMKII f/f/Rosa26-LacZ ⫹/⫺, nestin-Cre ERT2⫺/␣CaMKII f/⫹/Rosa26-LacZ ⫹/⫺, and nestin-Cre ERT2⫺/␣-CaMKII ⫹/⫹/
Rosa26-LacZ ⫹/⫺ offspring, of which only the nestin-Cre ERT2⫹ pups
were used. In the morphological and behavioral experiments, we compared nestin-Cre ERT2⫺/␣-CaMKII f/f/Rosa26-LacZ ⫹/⫺ (referred to as
␣-CaMKII f/f for simplicity) and nestin-Cre ERT2⫹/␣-CaMKII f/f/Rosa26LacZ ⫹/⫺ (referred to as ␣-CaMKII inducible knock-out or ␣-CaMKIIiKO) mice. For the proliferation, survival, and maturation analyses, we
compared LacZ ⫹ dentate granule cells in nestin-Cre ERT2⫹/␣-CaMKII ⫹/⫹/
Rosa26-LacZ ⫹/⫺ (referred to as ␣-CaMKII ⫹/⫹ mice for simplicity) to
LacZ ⫹ dentate granule cells in nestin-Cre ERT2⫹/␣-CaMKII f/f/Rosa26LacZ ⫹/⫺ (␣-CaMKII-iKO) mice. All mice were treated with TAM. Importantly, this design ensures that group effects cannot be attributed tononspecific effects of TAM.

Mice were bred in our colony at The Hospital for Sick Children andmaintained on a 12 h light/dark cycle with free access to food and water.
Before all behavioral experiments, mice were handled for 2 min twice a
day for 4 d. Male and female offspring were used in all experiments. All
experiments were performed in the light cycle and conducted according
to protocols approved by The Hospital for Sick Children Animal Careand Use Committee.

Drugs
TAM treatment. TAM (Sigma) was dissolved in ethanol (10%) and suspended in sunflower seed oil (Lagace et al., 2007). Mice received daily
injections (180 mg/kg; i.p.) for 5 d. Mice were treated with TAM either at
4 –5 or at 12 weeks of age.

Immunohistochemistry
Mice were perfused transcardially with PBS (0.1 M) and 4% PFA. Brains
were removed, fixed overnight in PFA, and transferred to 0.1 M PBS.
Coronal sections (40 ␮m) were cut using a vibratome (Leica VT1200S).
The following primary antibodies were used: mouse monoclonal anti-␣CaMKII (1:2000; Millipore), goat polyclonal anti-doublecortin (DCX)(1:4500; Santa Cruz Biotechnology), rabbit polyclonal anti-Egr1 (Zif268)(1:10,000; Santa Cruz Biotechnology), mouse monoclonal anti-NeuN(1:1000; Millipore), rabbit polyclonal anti-LacZ (1:6000; Invitrogen),mouse monoclonal anti-nestin (1:150; BD Biosciences PharMingen),rabbit monoclonal anti-calbindin (D28K; 1:600; Cell Signaling Technology), mouse monoclonal anti-calretinin (1:1500; Swant), rat monoclonalanti-BrdU (1:1000; Axyll), rabbit polyclonal anti-Ki67 (1:10,000; Abcam), anti-GFP (1:500; Invitrogen), and anti-VGlut1 (1:1000; Millipore). All sections were treated with 1% hydrogen peroxidase. Sections
were then incubated overnight with the primary antibody and then for 60
min at room temperature with HRP-conjugated or biotinylated secondary antibodies (1:750; Jackson ImmunoResearch Laboratories). Signals
were amplified and visualized using Vectastain Elite ABC kit (Vector
Laboratories), tyramide signal amplification, Alexa-Fluor-conjugatedstreptavidin (Invitrogen). Sections were mounted on slides with Permafluor anti-fade medium.

Imaging and quantification
All images were acquired using a confocal (LSM 710 Zeiss) microscope.
To calculate cell number, cell density, or proportion of double-positive
cells, we used 1/5 systematic section sampling fractions covering theentire anterior–posterior extent of the DG. To calculate the proportion of
double-labeled cells, confocal 1 ␮m Z-stack images were obtained using
ZEN software (Zeiss) with a minimal interval of 15 ␮m to prevent duplicate counts of the same cell. We estimated the total number of LacZ ⫹cells after TAM treatment using the optical fractionator method on an
Olympus BX61 epifluorescence microscope using a 60⫻, 1.45 NA objective and a motorized XYZ stage attached to a computer with Stereoinvestigator 9.1 (MBF Bioscience) (Chen et al., 2004). A random systematic
sampling was used for these stereological analyses (section interval of 1/5,
grid size of 250 ⫻ 250 ␮m, 2D counting frame of 90 ⫻ 90 ␮m using
fractionators of 30 ␮m in thickness). Tissue thickness measured in eachcounting frame was used to estimate the total number of LacZ ⫹ cells in
the entire DG. Conditions were optimized to obtain a Gundersen coefficient of error ⬍0.05 (Gundersen et al., 1999). We quantified BrdU ⫹cells throughout the entire anterior–posterior extent of the DG (11–13
sections per mouse, with every fifth section analyzed) using the Nikon
Eclipse 80i microscope. Granule cell layer volume was estimated in
␣-CaMKII ⫹/⫹ (n ⫽ 7) and ␣-CaMKII-iKO (n ⫽ 7) mice from 10 hippocampal slices per mouse (collected every sixth section) using the Cavalieri volume estimation method (magnification: 20⫻; grid spacing: 40
␮m; Cavalieri estimator module in Stereo Investigator, MBF Bioscience).

Retrovirus production and stereotaxic surgery
Retroviral labeling of new neurons. New neurons were labeled using apCAG-GFP replication-deficient retroviral vector (based on the Moloney murine leukemia virus) (Tashiro et al., 2006b). Retroviral vectors
were harvested from a stable producing cell line, and concentrated byultraspeed centrifugation (average of 3.5 ⫻ 10 9 IU/ml), as previously
described (Stone et al., 2011).

Arruda-Carvalho et al. • ␣-CaMKII and Hippocampal Neurogenesis

Stereotaxic surgery. Mice were treated with atropine sulfate (0.1 mg/kg,
i.p.) and anesthetized with chloral hydrate (400 mg/kg, i.p.) before being
placed in a stereotactic frame. The scalp was incised and retracted to drill
holes through the skull above the hippocampi. Viral vectors were injectedbilaterally into the DG (anteroposterior: ⫺2.2 mm; mediolateral: 1.8mm; and dorsoventral ⫺2.2 mm) via a glass micropipette (outer diameter, ⬃50 ␮m) connected to a Hamilton syringe. The injection volume(0.5 ␮l) and rate (0.1 ␮l/min) were controlled by an infusion pump
(SP100i; WPI). Micropipettes were left in place for an additional 10 min
to ensure complete diffusion of the virus.

Morphological analysis
Eight weeks after the viral injections, mice from all groups were transcardially perfused with PBS (0.1 M) and 4% PFA. The brains were postfixed
in 4% PFA at ⫺4°C until processed. Vibratome sections (50 ␮m) of the
hippocampus were collected and stored in PBS/glycerol/NaAzide solution until further processed.
Dendritic morphology. All images were acquired using a confocal (LSM
710 Zeiss) microscope. To characterize neuronal arborization confocal 2
␮m Z-stack images (40 objective) were obtained using ZEN software(Zeiss). Dendrites from GFP ⫹ granule cells (␣-CaMKII f/f, n ⫽ 118 dendrite segments from 5 mice; ␣-CaMKII-iKO, n ⫽ 81 dendrite segmentsfrom 8 mice) were traced with Neurolucida software (MBF Bioscience)by an experimenter blind to the experimental groups. Measures of dendritic complexity (number of branches and bifurcations, proportion ofneurons with single vs multiple primary dendrites) were batch-analyzed
with the LMeasure software (Scorcioni et al., 2008).
Spine and synapse analysis. High-resolution (1024 ⫻ 1024 pixels),
high-magnification (63⫻ objective) Z-stack images (0.45 ␮m step) wereacquired. Every stack was subjected to iterative deconvolution with inverse filtering (ImagePro Plus, MediaCybernetics) to deblur the VGlut1(red channel) and GFP (green channel) signals. For spine and synapse
analysis, a random sample of dendritic segments (length ⫽ 20 ␮m) of
GFP ⫹ cells (␣-CaMKII f/f, n ⫽ 41 dendrite segments from 4 mice;␣-CaMKII-iKO, n ⫽ 47 dendrite segments from 5 mice) was extracted.
Dendritic spines were defined as protrusions (length ⬍2 ␮m) stemmingfrom the dendritic shaft (spines analyzed: ␣-CaMKII f/f, n ⫽ 1360;
␣-CaMKII-iKO, n ⫽ 1489). Spine density was expressed as the numberof protrusions per 10 ␮m. Putative synapses were determined by thejuxtaposition of VGlut1 puncta on the terminal tip of the GFP-positive
dendritic spines. The proportion of spines directly apposed to VGlut1 ⫹puncta (as a proxy for functional synaptic terminals) was used to estimate the proportion of putative synapses.

General behavioral apparatus and procedures
In the fear conditioning experiments, context A (the training context)
consisted of a stainless steel conditioning chamber (31 cm ⫻ 24 cm ⫻ 21cm; Med Associates) with a stainless steel shock-grid floor. The grid floor
was positioned over a stainless-steel drop-pan, which was lightly cleaned
with 70% ethyl alcohol to provide a background odor. Shock grid bars
(diameter 3.2 mm) were spaced 7.9 mm apart. The front, top, and back of
the chamber were made of clear acrylic and the two sides made of modular aluminum. Mouse freezing behavior was monitored via overheadcameras. Freezing was assessed using an automated scoring system (Actimetrics), which digitized the video signal at 4 Hz and compared movement frame by frame to determine the amount of freezing.
During training, mice were placed in context A. In the weak training
experiment, mice were placed in the context and after 120 s presented
with a 2 s footshock (0.5 mA). Mice remained in the context for a further
60 s before being returned to their home cage. In the strong training
experiment, mice were placed in the context and presented with 2 sfootshocks (0.5 mA) at 120, 180, and 240 s. After the final footshock, miceremained in the context for an additional 60 s. Shock responsivity wasestimated by comparing mouse velocity in 2 s windows immediately
preceding versus during shock presentation using the following formula:(velocityshock ⫺ velocitypre-shock)/(velocityshock ⫹ velocitypre-shock).
Twenty-four hours after training, mice were placed back in the context
and freezing assessed in a 5 min test.

J. Neurosci., September 3, 2014 • 34(36):11919 –11928 • 11921

Specific experimental protocols

Characterization of ␣-CaMKII expression in dentate granule cells (DGCs).
Four- to 5-week-old WT C57BL/6 mice were perfused, and brain tissue
was processed for immunohistochemistry for ␣-CaMKII and different
markers (n ⫽ 5 or 6/group).
Characterization of efficiency of TAM-induced knock-out of ␣-CaMKII
in recombined cells. Four- to 5-week-old ␣-CaMKII-iKO mice (n ⫽ 5)
were treated with TAM. Eight weeks later, mice were perfused and brain
tissue was processed for immunohistochemistry for ␣-CaMKII and
LacZ.
Characterization of TAM-induced knock-out of ␣-CaMKII: proliferationand survival. Four- to 5-week-old ␣-CaMKII ⫹/⫹ and ␣-CaMKII-iKO mice
were treated with TAM. For the proliferation analysis, ␣-CaMKII ⫹/⫹ (n ⫽5) and ␣-CaMKII-iKO (n ⫽ 6) mice received a single injection of BrdU (100mg/kg) 24 h after the last TAM injection. They were then perfused 24 h later.
For the survival analysis, ␣-CaMKII ⫹/⫹ (n ⫽ 6) and ␣-CaMKII-iKO(n ⫽ 9) mice received a series of BrdU injections (100 mg/kg, 2 per day
for 3 d), starting 1 d after the last TAM injection. Eight weeks after thestart of TAM, mice were perfused and tissue was processed for
immunohistochemistry.
Characterization of TAM-induced knock-out of ␣-CaMKII: maturation
analysis. ␣-CaMKII ⫹/⫹ (n ⫽ 6 – 8/marker) and ␣-CaMKII-iKO (n ⫽6 –9/marker) mice were treated with TAM. Immunohistochemistry analysis of expression of LacZ and different markers occurred 8 weeks later.
Characterization of ␣-CaMKII expression in newly generated neurons.
To characterize the onset of ␣-CaMKII expression, 6-week-old WT mice(n ⫽ 3) were injected with BrdU (200 mg/kg) and perfused 1, 3, or 6
weeks after injection.
Characterization of TAM-induced knock-out of ␣-CaMKII: IEG expression. ␣-CaMKII-iKO mice (n ⫽ 4) were trained in context fear conditioning (see below). Ninety minutes after the completion of training,mice were perfused and tissue was processed for LacZ and Zif268 immunohistchemistry. Home cage ␣-CaMKII-iKO animals (n ⫽ 2) were perfused and processed at the same time.
Characterization of TAM-induced knock-out of ␣-CaMKII: dendritic
morphology and synapse analysis. ␣-CaMKII f/fand ␣-CaMKII-iKO mice
were treated with TAM. One week after the start of TAM, mice received
bilateral infusions of pCAG-GFP retrovirus into the DG. GFP ⫹ cells wereanalyzed 8 weeks later. Regardless of genotype, brain sections were
stained for GFP and LacZ, and in ␣-CaMKII-iKO mice only double
labeled cells were analyzed.
Open field. For open field testing, 8 weeks after TAM treatment,
␣-CaMKII f/f (n ⫽ 10) and ␣-CaMKII-iKO (n ⫽ 7) mice were placed in
the center of a square-shaped arena (45 cm ⫻ 45 cm ⫻ 20 cm height) andallowed to explore for 10 min. The open field apparatus was constructedof Plexiglas and was dimly lit from above. Mouse location was tracked bya camera located above the open field, and total distance traveled as wellas time spent in three different zones (outer, middle, inner) was measured (Limelight2, Actimetrics). Total distance traveled was used as ameasure of spontaneous motor activity, and distribution of activity indifferent regions of the arena was used as a measure of anxiety-related
behavior (Archer, 1973).
Forced swim test. To assess depressive-like behaviors, we used theforced swim test (Porsolt, 1979). ␣-CaMKII f/f (n ⫽ 8) and ␣-CaMKIIiKO (n ⫽ 14) mice were treated with TAM. Eight weeks later, mice wereplaced in a cylinder (radius, 6.5 cm, height 20 cm) for 6 min. The cylinder
was filled to a depth of 12 cm with 25 ⫾ 1°C water. The time spentmotionless was recorded for each mouse.
Contextual fear conditioning. ␣-CaMKII f/f and ␣-CaMKII-iKO mice
were treated with TAM. Eight weeks after the start of TAM treatment,
␣-CaMKII f/f (n ⫽ 10) and ␣-CaMKII-iKO (n ⫽ 9) mice were trainedwith a single footshock, and freezing was assessed in the training context
24 h later. A separate group of ␣-CaMKII f/f (n ⫽ 10) and ␣-CaMKII-iKO(n ⫽ 13) mice were trained with 3 footshocks, and tested 24 h later. For
the Zif268 quantification, ␣-CaMKII-iKO (n ⫽ 4) mice were trained with
5 footshocks (0.7 mAmp) and perfused 90 min later.

Arruda-Carvalho et al. • ␣-CaMKII and Hippocampal Neurogenesis

11922 • J. Neurosci., September 3, 2014 • 34(36):11919 –11928

Figure 1. Expression of ␣-CaMKII in dentate granule cells. Colocalization of ␣-CaMKII (green) expression with (A) nestin (red), (B) Ki67 (red), (C) DCX (red), (D) calretinin (red), (E) NeuN (red),and (F ) calbindin (red) in the dentate gyrus of 4- to 5-week-old mice. G–I, Time course of ␣-CaMKII expression. WT mice were injected with BrdU and killed 1, 3, or 6 weeks later. Examples of BrdU ⫹cells at (G) 1 week and (H ) 6 weeks. I, Percentage of BrdU ⫹ cells coexpressing ␣-CaMKII. Scale bar, 10 ␮m. Mo, Molecular layer; GCL, granule cell layer.
Table 1. ␣-CaMKII expression pattern in DGCs
Percentage of marker ⫹ cells expressing ␣CamKII (⫾ SEM)
Cells analyzed

NestinKI67

DCX

CalretNeuN

Calb

1.78 ⫾ 0.48564

0.85 ⫾ 0.41
609

18.4 ⫾ 2.7
1273

41.3 ⫾ 3.6
152

99.2 ⫾ 0.27
3264

99.4 ⫾ 0.19
2495

Data analysis

Cell counts and morphological analyses are displayed as mean ⫾ SEM.
Unless otherwise specified, data were analyzed using ANOVAs and t
tests. Between-group pairwise comparison of morphological features(number of branches and bifurcations) and spine density and synapse
number were performed by using the nonparametric Wilcoxon ranksum test because these datasets were not normally distributed.

Results

␣-CaMKII expression pattern in DGCs
We first examined ␣-CaMKII expression in maturing DGCs
in both the juvenile and adult mouse brain by staining forproteins expressed at different stages of cell differentiation(Kempermann et al., 2004). In 4- to 5-week-old WT mice,␣-CaMKII was rarely expressed in cells expressing progenitor

markers, such as nestin (Fig. 1A) or Ki67 (Fig. 1B). However,␣-CaMKII was expressed in ⬃20% (DCX) and 40% (calretinin) of cells expressing neuroblast markers (Fig. 1C,D), andin virtually all cells expressing mature neuronal markers, suchas NeuN and calbindin (Fig. 1 E, F; Table 1). We found an
equivalent pattern of expression in 12-week-old mice (datanot shown). In a separate experiment, 5- to 6-week-old mice
were injected with the proliferation marker BrdU, and␣-CaMKII expression was examined 1, 3, or 6 weeks later. In
the 1 week group, few BrdU ⫹ cells coexpressed ␣-CaMKII(Fig. 1G). However, the proportion of BrdU ⫹ cells expressing
␣-CaMKII increased markedly with the postinjection delay(Fig. 1 H, I ). Therefore, together these data indicate that
␣-CaMKII is expressed postmitotically, and the onset of ex-Arruda-Carvalho et al. • ␣-CaMKII and Hippocampal Neurogenesispression coincides with the late neuroblast stage (Nakashiba etal., 2012).
Conditional deletion of ␣-CaMKII fromadult-generated DGCs
To conditionally delete ␣-CaMKII from NPCs, we crossed mice expressing a TAM-inducible cre-recombinase driven by a progenitor
specific promoter (nestin-Cre ERT2 mice) (Imayoshi et al., 2008;
Arruda-Carvalho et al., 2011) with mice in which ␣-CaMKII isfloxed (␣-CaMKII f/f) (Achterberg et al., 2014). In double mutant
offspring from this cross (␣-CaMKII-iKO mice), TAM treatment
induces excision of the flanked sequences and deletion of ␣-CaMKII
in neural progenitor cells and their progeny (Fig. 2A). To visualize
recombined cells, ␣-CaMKII-iKO mice were additionally crossedwith a ROSA26-LacZ mouse line, in which a stop codon preventing
LacZ expression is flanked by lox P sites.
Eight weeks after TAM treatment, 56.4 ⫾ 6.7% of nestin ⫹cells in the DG expressed LacZ, indicating that TAM-induced
recombination was efficient, as previously observed (Imayoshi etal., 2008; Arruda-Carvalho et al., 2011). Stereological counts revealed that there were 30,912 ⫾ 2832 LacZ ⫹ cells in the DG.
These cells were uniformly distributed throughout the anterior–posterior extent of the DG, suggesting no intraregional differences in TAM-induced recombination efficiency. The vast
majority of the recombined (i.e., LacZ ⫹) cell population (92.2 ⫾
0.4%) lacked ␣-CaMKII (Fig. 2B), confirming the efficiency ofthe cre-recombinase dependent deletion.
Abnormal morphological development of ␣-CaMKIIdeficient, adult-generated DGCs
In WT mice, our immunohistochemical analyses indicated thatthe onset of ␣-CaMKII expression coincides with the late neuroblast stage, when the cells are ⬃3 weeks of age. This corresponds
to a period when adult-generated granule cells are becoming synaptically integrated into the entorhinal-DG-CA3 circuit (Zhao etal., 2006; Ge et al., 2007, 2012). Thereafter, dendritogenesis andspinogenesis/synaptogenesis continue, with structural changes
stabilizing after ⬃8 weeks (Zhao et al., 2006). We therefore asked
whether ␣-CaMKII deletion impacts dendritic developmentof adult-generated DGCs. To do this, ␣-CaMKII ⫹/⫹ and␣-CaMKII-iKO mice were treated with TAM, and then 1 week
later microinfused with a retrovirus expressing GFP into the DG(Stone et al., 2011). Retroviruses infect dividing neural progenitors and their progeny (Tashiro et al., 2006b). As GFP is expressed
throughout the cell body and processes of progeny, this strategy
can be used to conduct detailed morphological analysis of adultgenerated granule cells. Accordingly, 8 weeks after TAM treatment, dendritic morphology of infected granule cells was
examined in ␣-CaMKII f/f and ␣-CaMKII-iKO mice (Fig. 2C).
Infected cells in ␣-CaMKII-iKO mice had more complex dendritic morphology with a higher number of bifurcations (W ⫽3068, p ⬍ 0.0001) and branches (W ⫽ 3585, p ⬍ 0.005) (Fig.
2C–E). The proportion of infected cells with multiple versus single
primary dendrites did not differ between groups (␣-CaMKII f/f ⫽18%; ␣-CaMKII-iKO ⫽ 26.5%) (␹ 2 ⫽ 0.73; p ⫽ 0.39). These phenotypes in the 8-week-old knock-out cells are consistent with previous studies showing that ␣-CaMKII inhibition is associated withmore complex dendritic arborization (Wu and Cline, 1998; Zou andCline, 1999) and, overall, is consistent with a role for ␣-CaMKII in
activity-dependent structural stabilization.
In contrast to the morphological analyses, we found no differences in expression of proteins expressed at different stages ofdifferentiation in ␣-CaMKII ⫹/⫹ and ␣-CaMKII-iKO mice (Fig.

J. Neurosci., September 3, 2014 • 34(36):11919 –11928 • 11923

2F–I: [nestin: ␣-CaMKII ⫹/⫹ ⫽ 11.10 ⫾ 1.26, ␣-CaMKII-iKO ⫽13.76 ⫾ 1.86, t(14) ⫽ 1.27, p ⫽ 0.11; DCX: ␣-CaMKII ⫹/⫹ ⫽11.31 ⫾ 1.39, ␣-CaMKII-iKO ⫽ 14.63 ⫾ 1.64, t(14) ⫽ 1.54,
p ⫽ 0.07; NeuN: ␣-CaMKII ⫹/⫹ ⫽ 77.18 ⫾ 2.02, ␣-CaMKII-iKO ⫽79.04 ⫾ 4.61, t(15) ⫽ 0.38, p ⫽ 0.35; calbindin: ␣-CaMKII ⫹/⫹ ⫽82.72 ⫾ 1.72, ␣-CaMKII-iKO ⫽ 80.43 ⫾ 3.17, t(10) ⫽ 0.63,
p ⫽ 0.27]). Because the onset of ␣-CaMKII expression coincides
with the late neuroblast stage, the absence of differences at earlier
stages of differentiation is expected. Furthermore, equivalent expression of mature neuronal markers, such as NeuN and calbindin, indicates that ␣-CaMKII deficiency does not alter neuronalfate. Finally, recombined (LacZ ⫹) cells in ␣-CaMKII-iKO miceexpressed the activity-regulated gene Zif268 after behavioral testing (Fig. 2J ) (t(4) ⫽ 2.23, p ⬍ 0.05). This suggests that, althoughdendritic and synaptic structure was altered, ␣-CaMKII-deficient
cells respond to external stimulation.
We next examined whether deletion of ␣-CaMKII impacted
synaptic integration of newly generated granule cells into adulthippocampal circuits. To do this, in infected granule cells, wequantified the number of spines and the proportion of spines directly apposed to vesicular glutamate transporter 1 (VGlut1 ⫹)puncta (as a proxy for functional synaptic terminals) in ␣-CaMKII f/fand ␣-CaMKII-iKO mice. Eight weeks following TAM treatment,
spine density was not affected by ␣-CaMKII deletion (W ⫽ 1135,
p ⫽ 0.15) (Fig. 3A). However, in ␣-CaMKII-iKO mice, there wasa reduction in the proportion of spines in direct apposition to
VGlut1 ⫹ puncta (W ⫽ 1242, p ⫽ 0.02) (Fig. 3B). This suggeststhat there is a reduction in the proportion of functional synaptic
terminals onto ␣-CaMKII-deficient granule cells. Consistent
with previous data in the developing Xenopus (Wu et al., 1996),
these data indicate that ␣-CaMKII regulates synaptogenesis andsynaptic maturation in the adult brain.
Finally, we found that altered dendritic and synaptic maturation in ␣-CaMKII-deficient adult-generated granule cells was
associated with an overall reduction in survival. Control (␣CaMKII ⫹/⫹) and ␣-CaMKII-iKO mice were treated with TAM
and subsequently with BrdU to label dividing cells (Fig. 3C).
Eight weeks later, there was a reduction in number of BrdU ⫹ cellsin ␣-CaMKII-iKO brains (Fig. 3D) (t(13) ⫽ 2.41, p ⬍ 0.05). This
was not reflected in a difference in total granule cell layer
volume (t(12) ⫽ 1.009, p ⫽ 0.335; granule cell layer volume for␣-CaMKII ⫹/⫹ ⫽ 537.086 ⫾ 21.228 mm 3 ⫻ 10 ⫺3 and ␣-CaMKIIiKO ⫽ 574.628 ⫾ 30.834 mm 3 ⫻ 10 ⫺3). Because proliferation was
unaltered (data not shown), these data suggest that newly generated,
␣-CaMKII-deficient granule cells are less likely to survive, possibly
because of abnormal dendritic and synaptic maturation.
Impaired hippocampus-dependent learning in ␣-CaMKIIiKO mice
Deletion of ␣-CaMKII from progenitor cells led to altered granule cell morphology and a reduction in the number of putative
functional synaptic terminals. We therefore next asked whether
these mice exhibited impairments in hippocampus-dependent
learning. To do this, we trained ␣-CaMKII f/f and ␣-CaMKII-iKO
mice in a contextual fear conditioning task, in which mice learn
an association between a context and an aversive event (i.e., thedelivery of a mild footshock). When returned to the same context, contextual fear memory is inferred from an increase in freezing behavior (Kim and Fanselow, 1992) (Fig. 4A). Importantly,
several previous studies have shown that disruption of adult neurogenesis impairs learning in this task (Winocur et al., 2006;
Warner-Schmidt et al., 2008; Hernández-Rabaza et al., 2009; Ko
et al., 2009; Drew et al., 2010; Denny et al., 2012; but see Zhang et11924 • J. Neurosci., September 3, 2014 • 34(36):11919 –11928

Arruda-Carvalho et al. • ␣-CaMKII and Hippocampal Neurogenesis

Figure 2. Conditional deletion of ␣-CaMKII impairs morphological development of newly generated dentate granule cells. A, Schematic of the strategy for conditional deletion of ␣-CaMKII from
NPCs. In nestin-Cre ERT2⫹/␣-CaMKII f/f mice (␣-CaMKII-iKO), TAM administration leads to excision of ␣-CaMKII exon 2 flanked sequences and deletion of ␣-CaMKII in neural progenitor cells andtheir progeny. B, Eight weeks after TAM treatment, the vast majority of recombined cells (LacZ ⫹; red) lacked ␣-CaMKII (green). Scale bars, 10 ␮m. C, Retrovirally labeled dentate granule cells incontrol (left; ␣-CaMKII f/f) and ␣-CaMKII-iKO (right) mice, 8 weeks after infection. In ␣-CaMKII-iKO (right) mice, GFP-labeled dentate granule cells dendrites had more (D) bifurcations and (E)branches. Eight weeks after TAM treatment, expression of (F ) nestin (green), (G) DCX (green), (H ) NeuN (green), and (I ) calbindin (green) in recombined (LacZ ⫹; red) cells was similar in control (top;␣-CaMKII f/f) and ␣-CaMKII-iKO (bottom) mice. J, Left, Eight weeks after TAM treatment, expression of zif268 (green) in recombined (LacZ ⫹; red) in control (␣-CaMKII f/f) and ␣-CaMKII-iKO mice.
Right, Contextual fear conditioning (trained) induced zif268 expression in recombined (LacZ ⫹) cells in ␣-CaMKII-iKO mice compared with home cage (HC) controls. Scale bar, 10 ␮m. *p ⬍ 0.05.

Arruda-Carvalho et al. • ␣-CaMKII and Hippocampal Neurogenesis

J. Neurosci., September 3, 2014 • 34(36):11919 –11928 • 11925

Figure 3. Conditional deletion of ␣-CaMKII impairs integration and survival of newly generated dentate granule cells. A, Retrovirally labeled granule cell dendritic segments (green) in control(left; ␣-CaMKII f/f) and ␣-CaMKII-iKO (right) mice, 8 weeks after infection. Spine density was similar in ␣-CaMKII-iKO (closed bars) compared with ␣-CaMKII f/f (open bars) mice. Scale bar, 1 ␮m.
B, Retrovirally labeled granule cell spine terminals (green) and VGlut1 ⫹ puncta (red). Examples of nonjuxtaposed (top) and juxtaposed (bottom) spine terminals. In ␣-CaMKII-iKO mice, there wasa reduction in the proportion of spines in direct apposition to VGlut1 ⫹ puncta. Scale bar, 0.5 ␮m. C, ␣-CaMKII ⫹/⫹ and ␣-CaMKII-iKO mice were injected with TAM and subsequently injected with
BrdU to label newly generated neurons. BrdU was injected twice a day for 3 d, 24 h after the last TAM injection. D, Eight weeks later, the number of BrdU ⫹ cells (red) was reduced in ␣-CaMKII-iKO(open bars) compared with ␣-CaMKII ⫹/⫹ (closed bars) mice. Examples of BrdU ⫹ cells at higher magnification are shown from the insets (right). Scale bars: left, 100 ␮m; right, 10 ␮m. *p ⬍ 0.05.

al., 2008; Deng et al., 2009). During training, both ␣-CaMKII f/fand ␣-CaMKII-iKO mice exhibited similar reactivity to the footshock (t(17) ⫽ 0.75, p ⫽ 0.46) (Fig. 4B) and similar levels of
freezing before and after shock delivery (Genotype ⫻ Training
phase ANOVA, Training phase effect, F(1,18) ⫽ 14.65, p ⬍ 0.01;
no Genotype ⫻ Training phase interaction, F(1,18) ⫽ 0.39, p ⫽0.50). Planned comparisons indicated that there were no differencesin freezing levels in the postshock phase of training (t(18) ⫽ 0.80, p ⫽0.43) (Fig. 4C). These data indicate that the absence of ␣-CaMKII
did not alter nociception or interfere with the ability to freeze. However, when placed back into the same context 24 h later, ␣-CaMKIIiKO froze less than their ␣-CaMKII f/f littermates (Fig. 4D,E) (t(17) ⫽1.79, p ⬍ 0.05). These results indicate that ␣-CaMKII-iKO mice are
impaired in a form of one-trial fear learning.
A previous study found that suppression of adult neurogenesis similarly impaired acquisition of contextual fear conditioning under these weak, but not strong, training conditions (Drew
et al., 2010). To evaluate whether contextual fear conditioning
was impacted after strong training, we next trained ␣-CaMKII f/fand ␣-CaMKII-iKO with three shocks (Fig. 4F ). As before, during training shock reactivity and freezing levels were similar in
␣-CaMKII f/f and ␣-CaMKII-iKO mice (Genotype ⫻ Training
phase ANOVA, Training phase effect, F(3,22) ⫽ 63.55, p ⬍ 0.0001;

no Genotype ⫻ Training phase interaction, F(3,22) ⫽ 2.26, p ⫽0.09). Planned comparisons indicated that there were no differencesin shock reactivity (t(22) ⫽ 0.48, p ⫽ 0.64) or freezing levels in thepostshock phase of training (t(18) ⫽ 0.80, p ⫽ 0.43) (Fig. 4G,H).
However, in contrast to the weak training conditioning, following
strong training ␣-CaMKII f/f and ␣-CaMKII-iKO mice exhibited
equivalent levels of conditioned fear when tested 24 h later (t(21) ⫽0.56, p ⫽ 0.58) (Fig. 4I,J). These results indicate that stronger training may overcome learning deficits observed in ␣-CaMKII-iKO
mice, and confirm that conditional deletion of ␣-CaMKII from
NPCs does not interfere with the propensity to freeze.
Adult neurogenesis is implicated in regulation of mood/anxiety (Sahay and Hen, 2007). To evaluate whether emotionality
was altered, we tested separate groups of ␣-CaMKII f/f and␣-CaMKII-iKO mice in the open field and forced swim tests. In the
open field, total locomotor activity was similar in ␣-CaMKII f/f and␣-CaMKII-iKO mice, suggesting that exploratory behavior is unaffected (t(15) ⫽ 0.31, p ⫽ 0.76) (Fig. 4K ). Furthermore, ␣-CaMKII f/fand ␣-CaMKII-iKO mice spent equivalently little time in thecenter of the open field, suggesting that anxiety is unaltered (twoway ANOVA; effect of zone only F(2,30) ⫽ 64.60, p ⬍ 0.0001) (Fig.
4L). In the forced swim test, latency to become immobile (t(22) ⫽0.94, p ⫽ 0.36) and total time immobile (t(22) ⫽ 0.71, p ⫽ 0.48) was

11926 • J. Neurosci., September 3, 2014 • 34(36):11919 –11928

Arruda-Carvalho et al. • ␣-CaMKII and Hippocampal Neurogenesis

Figure 4. Conditional deletion of ␣-CaMKII impairs contextual fear learning. A, Experimental design. Mice underwent contextual fear conditioning (1 shock) 8 weeks after TAM administration
and were tested 1 d later. During training, (B) shock reactivity and (C) postshock freezing were similar in control (␣-CaMKII f/f; closed bars) and ␣-CaMKII-iKO (open bars) mice. During testing,␣-CaMKII-iKO showed reduced freezing levels (D, minute-by-minute freezing; E, total freezing in the 5 min test). F, Experimental design. Mice underwent contextual fear conditioning (3 shocks) 8
weeks after TAM administration and were tested 1 d later. During training, (G) shock reactivity and (H ) postshock freezing were similar in control (␣-CaMKII f/f; closed bars) and ␣-CaMKII-iKO (openbars)mice.Duringtesting, ␣-CaMKII f/f and ␣-CaMKII-iKOmiceexhibitedequivalentfreezinglevels(I,minute-by-minutefreezing;J,totalfreezinginthe5mintest).Intheopenfield, ␣-CaMKII f/f (closedbars)and ␣-CaMKII-iKO (open bars) mice (K) covered equivalent total distance and (L) spent equivalent time in different regions of the arena. In the forced swim test, (M) latency to become immobile and (N) total
time spent immobile were equivalent in ␣-CaMKII f/f (closed bars) and ␣-CaMKII-iKO (open bars) mice. *p ⬍ 0.05.

equivalent in ␣-CaMKII f/f and ␣-CaMKII-iKO mice (Fig. 4M,N),suggesting no differences in basal levels of depressive-like behaviors.
These data further suggest that that ␣-CaMKII deficiency in newly
generated granule cells does not lead to a general alteration in
behavior.

Discussion
Here we developed a transgenic strategy to conditionally delete
␣-CaMKII from neural progenitor cells and their progeny, andstudied the cell-autonomous role of ␣-CaMKII in morphologicaland synaptic development in the adult hippocampus. There were
three primary findings. First, selective deletion of ␣-CaMKII fromnewly generated dentate granule cells led to an increase in dendritic
complexity. Second, ␣-CaMKII deletion led to a reduction in number of mature synapses and cell survival. Third, consistent with altered dendritic and synaptic development, acquisition of one-trial
contextual fear conditioning was impaired in ␣-CaMKII-iKO mice.

Previous work in C. elegans (Rongo and Kaplan, 1999), Drosophila(Wang et al., 1994), Xenopus (Wu et al., 1996; Zou and Cline, 1999;
Wu and Cline, 1998), and mouse (Pattinson et al., 2006) identified
␣-CaMKII as playing a key role in the stabilization of dendritic andsynaptic structure in the developing nervous system. The current
study indicates ␣-CaMKII plays a similar, cell-autonomous role in
the adult hippocampus, and, in addition, reveals that the loss of
␣-CaMKII from adult-generated granule cells is associated with impaired hippocampus-dependent learning.
The vast majority of cells generated in the subgranular zone of
the hippocampus differentiate into dentate granule cells, extending apical dendrites into the molecular layer of the hippocampus
and mossy fiber axons toward the CA3 region. However, it is not
until ⬃2.5 weeks that these cells form input and output synaptic
connections, a time point that coincides with the onset of
␣-CaMKII expression. Thereafter continues a period of contin-

Arruda-Carvalho et al. • ␣-CaMKII and Hippocampal Neurogenesis

ued growth and eventually stabilization of dendritic and synaptic
structures, where connections are likely refined in an activitydependent manner (Tronel et al., 2010). In the developing nervous system, ␣-CaMKII has been shown to play a critical role inthis refinement process, acting, for example, to attenuate dendritogenesis and stabilize dendritic structure. Our data here suggestthat this role is conserved as newly generated granule cells integrate
into adult hippocampal circuits. We found that ␣-CaMKIIdeficient, newly generated granule cells exhibited increased dendritic
complexity (more dendritic bifurcations and branches), consistent
with the idea that ␣-CaMKII acts as a brake on continued dendritogenesis (Cline, 2001). Additionally, there was a reduction in
number of mature synapses on ␣-CaMKII deficient granule cells,consistent with a role for ␣-CaMKII in promoting synapse maturation. In our study, ␣-CaMKII deletion was limited to NPCs
and their progeny and did not impact postmitotic neuronal populations. Therefore, our data additionally indicate that ␣-CaMKII regulates these processes in a cell-autonomous manner.
The refinement of neural circuits during development is regulated in an activity-dependent manner (Katz and Shatz, 1996),
with synaptic strengthening leading to the stabilization of dendritic structure (Cline, 2001). Because ␣-CaMKII plays an essential role in synaptic strengthening, it is thought to provide anessential link between local activity levels and the stabilization ofdendritic structure, and it is likely that ␣-CaMKII plays a similarrole in the integration of adult-generated granule cells into adulthippocampal circuits. Coordinated presynaptic and postsynaptic
activity can lead to long-lasting strengthening of synaptic connections through LTP. During LTP, opening of NMDARs leads
to an increase in intracellular calcium levels that activates
␣-CaMKII (Lisman et al., 2012; Hell, 2014). Once activated,
␣-CaMKII translocates to the synapse (Shen and Meyer, 1999),
binds to NMDARs (Barria and Malinow, 2005), and strengthens
synaptic transmission through a signaling cascade that increases
AMPA receptor number (Shi et al., 1999; Hayashi et al., 2000)
and conductance (Derkach et al., 1999; Poncer et al., 2002; Lüthi
et al., 2004). NMDAR- ␣-CaMKII complexes are necessary for
LTP (Barria and Malinow, 2005) and can be persistent once
formed (Bayer et al., 2006). Consistent with the idea that NMDA␣-CaMKII signaling plays an essential role in stabilization of dendritic arborization and synapse maturation, the conditionaldeletion of the NMDAR subunit NR1 from NPCs during adulthood leads to an increase in cell death during a critical periodmarked by high synaptogenesis and the onset of ␣-CaMKII expression (Tashiro et al., 2006a). The fact that deletion of
␣-CaMKII in a similar cell population also leads to a decrease insurvival supports the hypothesis that activity, potentially througha NMDAR-␣-CaMKII pathway, is a crucial survival signal to shapecircuits in the continuously evolving DG. Similarly, conditional deletion of the NMDAR subunit NR2B from NPCs during adulthood
also disturbed dendritic morphology in the DG and was associatedwith hippocampal learning deficits (Kheirbek et al., 2012).
Our results suggest that one consequence of perturbing normal integration of newly generated granule cells into hippocampal circuits is impaired hippocampus-dependent learning. The
transgenic approach we used allowed for efficient deletion of
␣-CaMKII from NPCs and their progeny. Nonetheless, 8 weeks
after TAM treatment, we estimated that the population of
␣-CaMKII-deficient newly generated granule cells to be⬃30,000. As the mouse adult DG contains ⬃500,000 granule cells(Ihunwo and Schliebs, 2010; Arruda-Carvalho et al., 2011), this
subpopulation of ␣-CaMKII-deficient granule cells represents

J. Neurosci., September 3, 2014 • 34(36):11919 –11928 • 11927

only ⬃6% of the total granule cell population. Therefore,
␣-CaMKII deficiency in a relatively small proportion of cells is
sufficient to produce deficits in hippocampus-dependent learning. Although this may be surprising, similar results have been
observed following NPC-specific deletion of other genes using
either transgenic (Zhang et al., 2008; Guo et al., 2011; Kheirbek etal., 2012; Pan et al., 2012; Cancino et al., 2013) or even viral (Zhouet al., 2013) strategies, which would impact far fewer adultgenerated granule cells. Because the integration of newborn cells
into the hippocampus is a fine-tuned process, it is possible thateven small perturbations of this process can impact hippocampal
function. It is likely that ␣-CaMKII deficiency not only compromises anatomical integration but also impairs plasticity of thosecells once integrated. Therefore, an alternative (but not necessarily mutually exclusive) possibility is that learning deficits are related to the reduced potential for strengthening of synaptic inputs
onto ␣-CaMKII-deficient cells during conditioning.

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